Mitigating tissue damage and fibrosis via latent transforming growth factor beta binding protein (ltbp4)

ABSTRACT

The disclosure relates to compositions and methods of mitigating tissue damage and fibrosis in a patient by modulating latent transforming growth factor beta binding protein (LTBP4)-induced proteolysis of a TGFβ superfamily protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/857,122, filed Dec. 28, 2017, which is a Continuation of U.S.application Ser. No. 13/957,100, filed Aug. 1, 2013, which claims thepriority benefit under 35 U.S.C. § 119(e) of Provisional U.S. PatentApplication No. 61/678,564, filed Aug. 1, 2012, the disclosure of whichis incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant NumberHL61322, awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

FIELD

The disclosure relates to compositions and methods of mitigating tissuedamage and fibrosis in a patient via latent transforming growth factorbeta binding protein (LTBP4).

SEQUENCE LISTING

This application contains, as a separate part of the disclosure, aSequence Listing in computer-readable form (filename:46577C_SeqListing.txt; created: Sep. 5, 2018; 220,906 bytes—ASCII textfile) which is incorporated by reference in its entirety.

BACKGROUND

The transforming growth factor (TGF) beta superfamily proteins are keyregulators of fibrosis in all parenchymal organs [Kisseleva et al., ProcAm Thorac Soc. 5: 338-42 (2008)]. Duchenne Muscular Dystrophy (DMD) ischaracterized by progressive fibrosis that is accompanied by increasedTGFβ signaling [Bernasconi et al., J Clin Invest. 96: 1137-44 (1995);Chen et al., Neurology 65: 826-34 (2005)]. In DMD, fibrosis not onlycontributes directly to muscle dysfunction but also inhibitsregeneration. DMD is characterized by muscle membrane fragility thatleads to progressive myofiber loss. With disease progression, DMD muscleis replaced by fibrosis. Although muscle is highly regenerative,regeneration in DMD is not sufficient to offset degeneration leading tomuscle weakness. Glucocorticoid steroids are used to slow progression inDMD, but use of steroids is complicated by side effects includingosteoporosis and weight gain (Bushby et al., 2010). Experimentaltherapies for DMD include approaches to increase dystrophin expression,modulate the inflammatory response, promote muscle growth and reducefibrosis [Bushby et al., Lancet 374: 1849-56 (2009)].

In recent years, biological compounds such as antibodies have shownefficacy for treating chronic diseases. For example, antibodies directedagainst TNFα (infliximab) or anti-TNF receptor (etanercept) are now inwide use for rheumatoid arthritis and other related disorders. Whileinitially developed for its anti-cancer activity, the anti-VEGF antibodyis now used to treat macular degeneration (bevacizumab). Thus, long-termuse with biological compounds can be effective and safe. Consistent withtherapeutic approaches comprising the administration of a biologicalcompound such as an antibody is the fact that antibodies are readilydetected in the matrix of dystrophic muscle, such as the muscle of DMDpatients.

A number of approaches, including but not limited to angiotensininhibition, either through the converting enzyme or the angiotensinreceptor, aldosterone inhibition, and inhibition by antibodies directedagainst TGFβ have been or are being tested to reduce fibrosis in DMD.[Cohn et al., Nat Med. 13: 204-10 (2007); Rafael-Fortney et al.,Circulation. 124: 582-8 (2011); Nelson et al., Am J Pathol. 178: 2611-21(2011)]. A major limitation of these approaches is that these drugs aresystemically active and often have unwanted effects such as reducedblood pressure. Given the relative hypotension of DMD patients,especially advanced DMD patients, such approaches are limited.

SUMMARY

Disclosed herein are compositions and methods for treating atransforming growth factor beta superfamily protein-related disease.Compositions according to the disclosure modulate the activity and/orproteolysis of latent TGFβ binding protein 4 (LTBP4). Methods accordingto the disclosure comprise administration of an effective amount of amodulator of LTBP4, with that effective amount being an amountsufficient to prevent, delay onset and/or treat a disorder according tothe disclosure. The compositions and methods provided by the disclosurewill improve one or more symptoms associated with disorders according tothe disclosure in afflicted individuals, thereby improving their qualityof life while alleviating the financial, psychological and physicalburdens imposed on modern healthcare systems.

Accordingly, in one aspect the disclosure provides a method of treatinga patient having a transforming growth factor beta (TGFβ) superfamilyprotein-related disease, comprising administering an effective amount ofan agent that modulates proteolysis of latent TGFβ binding protein 4(LTBP4) to a patient in need thereof.

A related aspect of the disclosure provides methods of delaying onset orpreventing a transforming growth factor beta (TGFβ) superfamilyprotein-related disease, comprising administering an effective amount ofan agent that modulates proteolysis of latent TGFβ binding protein 4(LTBP4) to a patient in need thereof.

In various embodiments of the foregoing methods, the patient has adisease selected from the group consisting of Duchenne MuscularDystrophy, Limb Girdle Muscular Dystrophy, Becker Muscular Dystrophy,myopathy, cystic fibrosis, pulmonary fibrosis, cardiomyopathy, acutelung injury, acute muscle injury, acute myocardial injury,radiation-induced injury and colon cancer.

In further embodiments, the agent is selected from the group consistingof an antibody, an inhibitory nucleic acid and a peptide.

In further aspects of the disclosure, the methods disclosed hereinfurther comprise administering an effective amount of a second agent,wherein the second agent is selected from the group consisting of amodulator of an inflammatory response, a promoter of muscle growth, achemotherapeutic agent and a modulator of fibrosis.

Another aspect of the disclosure is drawn to a method of treating apatient having a transforming growth factor beta (TGFβ)-related disease,comprising administering to the patient an effective amount of an agentthat upregulates the activity of latent TGFβ binding protein 4 (LTBP4).

A further aspect of the disclosure provides a method of delaying onsetor preventing a transforming growth factor beta (TGFβ)-related disease,comprising administering to the patient an effective amount of an agentthat upregulates the activity of latent TGFβ binding protein 4 (LTBP4).

In some embodiments of the methods, LTBP4 interacts with a TGFβsuperfamily protein, and in still further embodiments the TGFβsuperfamily protein is selected from the group consisting of TGFβ, agrowth and differentiation factor (GDF), activin, inhibin, and a bonemorphogenetic protein. In specific embodiments, the GDF is myostatin.

In additional embodiments, the agent is selected from the groupconsisting of a peptide, an antibody and a polynucleotide capable ofexpressing a protein having LTBP4 activity, each as disclosed herein. Insome embodiments, the polynucleotide is contained in a vector and infurther embodiments the vector is a viral vector. The disclosure furthercontemplates embodiments wherein the viral vector is selected from thegroup consisting of a herpes virus vector, an adeno-associated virus(AAV) vector, an adeno virus vector, and a lentiviral vector. In oneembodiment, the AAV vector is recombinant AAV9.

In some embodiments, the compositions and methods disclosed herein arefor treating a transforming growth factor beta-related disease in apatient. In particular embodiments, the patient suffers from a diseaseselected from the group consisting of Duchenne Muscular Dystrophy (DMD),Limb Girdle Muscular Dystrophy (LGMD), Becker Muscular Dystrophy (BMD),myopathy, cystic fibrosis, pulmonary fibrosis, cardiomyopathy, acutelung injury, acute muscle injury, acute myocardial injury,radiation-induced injury and colon cancer.

An additional aspect of the disclosure is drawn to methods as disclosedabove that further comprise administering a therapeutically effectiveamount of a second agent that is selected from the group consisting of amodulator of an inflammatory response, a promoter of muscle growth, achemotherapeutic agent and a modulator of fibrosis.

In some embodiments, an isolated antibody is provided that specificallybinds to a peptide comprising any one of the sequences set forth in SEQID NOs: 2-5. In further embodiments, the disclosure provides an isolatedantibody that specifically binds to a peptide that is at least 70%identical to a peptide comprising any one of the sequences set forth inSEQ ID NOs: 2-5, wherein the antibody retains an ability to specificallybind to LTBP4 and to decrease the susceptibility of LTBP4 toproteolysis.

Still further embodiments of the disclosure provide a peptide comprisingthe sequence as set out in SEQ ID NOs: 2-5, or a peptide that is atleast 70% identical to any one of the sequences as set out in SEQ ID NO:2-5 that retains an ability to act as a substrate for a protease.

In another aspect, a pharmaceutical formulation is provided comprisingan effective amount, such as a therapeutically effective amount, of anantibody and/or peptide of the disclosure, and a pharmaceuticallyacceptable carrier or diluent.

A further aspect of the disclosure provides a kit comprising aneffective amount, such as a therapeutically effective amount, of anantibody and/or peptide of the disclosure, a pharmaceutically acceptablecarrier or diluent and instructions for use.

In some embodiments, the formulation or the kit of the disclosurefurther comprises an effective amount, such as a therapeuticallyeffective amount, of a second agent, wherein the second agent isselected from the group consisting of a modulator of an inflammatoryresponse, a promoter of muscle growth, a chemotherapeutic agent and amodulator of fibrosis.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, because various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a model for action of LTBP4 (latent TGFβ binding protein4). LTBP4 binds directly to TGFβ family member proteins. In theextracellular matrix, the complex of LTBP4 protein and TGFβ forms thelarge latent complex. With proteolysis, LTBP4 undergoes a conformationalchange which releases TGFβ, thereby making it available for release andbinding TGFβ receptors on neighboring cells. TGFβ binding to itsreceptor results in TGFβ signaling in cells.

FIG. 2 depicts the gene structure of LTBP4. An insertion deletionpolymorphism in Ltbp4 alters the proline-rich region in mice. TheN-terminus of LTBP binds the extracellular matrix (ECM). The LTBP4protein is composed of multiple epidermal growth factor (EGF) repeatsinterspersed with motifs containing 8 cysteine residues (8-Cys). Thethird 8-cys repeat binds TGFβ directly. The proline-rich region (labeledhorizontal rectangle) separates the matrix-binding domain from theremainder of the protein. Mouse129 is protected against musculardystrophy because of insertion of 12 amino acids in the proline-richregion. Muscular dystrophy in D2 strains of mice is more severe. Rat,dog, cow, and humans each harbor a larger deletion of the proline-richregion of LTBP4.

FIG. 3 depicts results of studies using fragments of human and mouseLTBP4 that were expressed and digested. Human LTBP4 is more readilycleaved than murine LTBP4. The amino acid positions indicated for TP andTP2E are based on the human isoform a LTBP4 sequence (SEQ ID NO: 1).

FIG. 4 depicts results of studies using a blocking antibody that wasdesigned to recognize and bind the proline-rich region (Y) of LTBP4.When incubated with cell lysates expressing LTBP4, the presence of theantibody inhibits cleavage by plasmin. A nonspecific antibody did notinhibit cleavage.

FIG. 5 depicts that the proline-rich region of human LTBP4 is moreeasily cleaved than murine LTBP4.

FIG. 6 shows results of a study using a blocking antibody that inhibitedcleavage of full-length LTBP4. A nonspecific blocking antibody showed noeffect. Assays were conducted in triplicate with significant inhibitionof proteolysis observed.

FIG. 7 shows results of a study wherein a bacterial artificial transgeneexpressing human LTBP4 (hLTBP4 Tg) was crossed into the mouse mdx modelof Duchenne Muscular Dystrophy. (A) hLTBP4/mdx mice have enhancedfibrosis in their muscles as determined grossly through histology and bydirect quantitation. When quantified, fibrotic area was increased inhLTBP4/mdx mice compared to littermate mdx mice. (B) hLTBP4/mdx micehave reduced grip strength. Grip strength was compared betweenhLTBP4/mdx mice and mdx mice to determine whether the human LTBP4 geneworsens the muscle disease seen in mdx mice. hLTBP4/mdx mice are weakerthan mdx littermates (*). Grip strength was measured using the Treat NMDstandard protocols.

FIG. 8 shows that LTBP4 forms a complex with myostatin. HEK293 cellswere transfected with LTBP4 and epitope-tagged myostatin. LTBP4 wasprecipitated with two different anti-LTBP4 antibodies (lanes 3 and 5),and the precipitate was then immunoblotted with anti-myc antibody.Unprocessed myostatin was detected in the immunoprecipitate (arrow). Theupper band in lanes 1 and 2 that migrates above 50 KDa is endogenousc-myc, which is 63 KDa.

FIG. 9 depicts the results of experiments testing the effects ofcardiotoxin on both wild-type mice and transgenic mice that expresshuman LTBP4. A) transgenic mice displayed enhanced injury aftercardiotoxin injury seen as greater inflammatory mononuclear cellinfiltrate and fibrosis and fat deposition into the injured muscle. B)LTBP4 protein levels are increased after injury.

FIG. 10 shows that anti-LTBP4 antibodies mitigate muscle injury in vivo.Compared to PBS-injected mice, LTBP4-831 antibody-treated mice showedreduced central nucleation (panel A) and reduced fibrosis (panel B)following cardiotoxin injection.

FIG. 11 shows that increased TGFβ signaling is associated with increasedmacrophage infiltration in hLTBP4/mdx muscle compared to mdx muscle. A)Muscles were stained with antibodies to activated macrophages using theF4/80 antibody. B) hLTBP4/mdx muscle shows an increase in cleaved LTBP4protein compared to mdx, while little LTBP4 protein is seen in wild typeand hLTBP4 muscle in the absence of injury or muscular dystrophy. C)Proteolytic cleavage and a conformational change in LTBP4 is associatedwith TGFβ release.

DETAILED DESCRIPTION

The transforming growth factor beta (TGFβ) superfamily consists of morethan 40 members including TGFβ, activins, inhibins, growthdifferentiation factors and bone morphogenetic proteins (BMPs). Allmembers of this family share common sequence elements and structuralmotifs. They are multifunctional regulators of cell division,differentiation, migration, adhesion, organization and death, promotingextracellular matrix (ECM) production, tissue homeostasis andembryogenesis [Massague et al., Genes Dev 19: 2783-810 (2005); Javelaudet al., Int J Biochem Cell Biol 36: 1161-5 (2004); Moustakas et al.,Immunol Lett 82: 85-91 (2002)]. Among these proteins, TGFβ has a crucialrole in tissue homeostasis and the disruption of the TGFβ pathway hasbeen implicated in many human diseases, including cancer, autoimmune,fibrotic, and cardiovascular diseases [Ruiz-Ortega et al.,Cardiovascular Research 74: 196-206 (2007)].

TGFβ is synthesized as an inactive protein, named latent TGFβ, whichconsists of a main region and a latency associated peptide (LAP). Thisprotein interacts with the latent TGFβ binding proteins (e.g., LTBP4)and is anchored in the extracellular matrix (ECM). TGFβ is activatedfollowing proteolysis of LTBP4, which results in release of TGFβ.Specifically, and as disclosed herein, the proline-rich region of LTBP4is susceptible to proteolysis by a protease, and this proteolysis leadsto release and activation of TGFβ.

Active TGFβ then binds its receptors and functions in autocrine andparacrine manners to exert its biological and pathological activitiesvia Smad-dependent and independent signaling pathways [Lan, Int J BiolSci 7(7): 1056-1067 (2011); Derynck et al., Nature. 425: 577-84 (2003)].

Thus, inhibition of the proteolysis of LTBP4 will inhibit the release ofbound TGFβ, and the resulting sequestration of TGFβ will inhibit thedownstream signaling effects of TGFβ, resulting in mitigation ofTGFβ-related disease.

The working examples and experimental data disclosed therein demonstratethat the proline-rich region of LTBP4 is susceptible to proteolysis.These results support therapeutics and therapies directed to modulatingthe proteolysis of LTBP4 in a patient having a TGFβ-related disease.

The experimental results disclosed herein also demonstrate thatproteolysis of LTBP4 can be inhibited by antibodies. Inhibition of LTBP4proteolysis using pharmacological approaches is expected to provide aneffective approach to the treatment of TGFβ-related diseases.

Experimental results disclosed herein additionally demonstrate that afragment of human LTBP4 is more susceptible to proteolysis than themouse LTBP4 sequence. Consequently, a phenomenon elucidated in the mouseis mirrored in humans, and inhibition of LTPB4 proteolysis is expectedto provide an effective treatment for TGFβ-related diseases.

Unless otherwise defined herein, scientific and technical terms employedin the disclosure shall have the meanings that are commonly understoodand used by one of ordinary skill in the art. Unless otherwise requiredby context, it will be understood that singular terms shall includeplural forms of the same and plural terms shall include the singular.Specifically, as used herein and in the claims, the singular forms “a”and “an” include the plural reference unless the context clearlyindicates otherwise.

As used in the disclosure, the term “treating” or “treatment” refers toan intervention performed with the intention of preventing the furtherdevelopment of or altering the pathology of a disease or infection.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Of course, when “treatment” isused in conjunction with a form of the separate term “prophylaxis,” itis understood that “treatment” refers to the narrower meaning ofaltering the pathology of a disease or condition. “Preventing” refers toa preventative measure taken with a subject not having a condition ordisease. A therapeutic agent may directly decrease the pathology of adisease, or render the disease more susceptible to treatment by anothertherapeutic agent(s) or, for example, the host's immune system.Treatment of patients suffering from clinical, biochemical, orsubjective symptoms of a disease may include alleviating one or more ofsuch symptoms or reducing the predisposition to the disease. Improvementafter treatment may be manifested as a decrease or elimination of one ormore of such symptoms.

As used herein, the phrase “effective amount” is meant to refer to anamount of a therapeutic (i.e., a therapeutically effective amount),prophylactic (i.e., a prophylactically effective amount), orsymptom-mitigating (i.e., a symptom-mitigating effective amount)compound (e.g., agent or second agent) sufficient to modulateproteolysis of latent TGFβ binding protein 4 (LTBP4), such as would beappropriate for an embodiment of the disclosure in eliciting the desiredtherapeutic, prophylactic, or symptom-mitigating effect or response,including alleviating one or more of such symptoms of disease orreducing the predisposition to the disease.

As used herein, “hybridization” means the pairing of substantiallycomplementary strands of polymeric compounds. One mechanism of pairinginvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleotidebases (nucleotides) of the strands of polymeric compounds. For example,adenine and thymine are complementary nucleotides which pair through theformation of hydrogen bonds. Hybridization can occur under varyingcircumstances.

An antisense compound is “specifically hybridizable” when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a modulation of function and/oractivity, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target nucleicacid sequences under conditions in which specific binding is desired,i.e., under physiological conditions in the case of in vivo applicationssuch as therapeutic treatment, and under conditions in which assays areperformed in the case of in vitro assays.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a compound(e.g., agent) disclosed herein will hybridize to its target sequence,but to a minimal number of other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances andin the context of this disclosure, “stringent conditions” under whichpolymeric compounds hybridize to a target sequence are determined by thenature and composition of the polymeric compounds and by theapplication(s) involved. In general, stringent hybridization conditionscomprise low concentrations (<0.15M) of salts with inorganic cationssuch as Na⁺⁺ or K⁺⁺ (i.e., low ionic strength), temperatures higher than20° C.-25° C. below the T_(m) of the polymeric compound:target sequencecomplex, and the presence of denaturants such as formamide,dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecylsulfate (SDS). An example of a set of high stringency hybridizationconditions is 0.1× sodium chloride-sodium citrate buffer (SSC)/0.1%(w/v) SDS at 60° C. for 30 minutes.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides on one or two polymeric strands.Consistent with Watson-Crick base pairing rules (A binds T or U; G bindsC; where A, G, C, T and U are the conventional ribo-, or deoxyribo-,nucleotide monophosphates). “Specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof precise nucleotide pairing or complementarity over a sufficientnumber of nucleotides such that stable and specific binding occursbetween the polymeric compound and a target nucleic acid. The terms thusallow for base pairing gaps, but not to the extent that it preventsstable and specific binding.

It is understood in the art that the sequence of a polymeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, a polynucleotide may hybridize overone or more segments such that intervening or adjacent segments are notinvolved in the hybridization event (e.g., a loop structure, mismatch orhairpin structure). The polymeric compounds of the present disclosurecomprise at least about 70%, or at least about 75%, or at least about80%, or at least about 85%, or at least about 90%, or at least about95%, or at least about 99% sequence complementarity to a target region,within the target nucleic acid sequence to which they are targeted. Forexample, an antisense compound in which 18 of 20 nucleotides of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleotides may be clustered or interspersed with complementarynucleotides and need not be contiguous to each other or to complementarynucleotides. As such, an antisense compound which is 18 nucleotides inlength having 4 (four) noncomplementary nucleotides which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present disclosure. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined by use of routine sequence comparisonsoftware and algorithms, e.g., BLAST programs (basic local alignmentsearch tools) and PowerBLAST programs known in the art [Altschul et al.,J. Mol. Biol., 215: 403-410 (1990); Zhang and Madden, Genome Res., 7:649-656 (1997)]. Percent homology, sequence identity or complementarity,can be determined by, for example, the Gap program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.), using default settings, whichuses the algorithm of Smith and Waterman [Adv. Appl. Math., 2: 482-489(1981)].

As used herein, the term “(T_(m))” means melting temperature and refersto the temperature, under defined ionic strength, pH, and nucleic addconcentration, at which 50% of the polynucleotides complementary to thetarget sequence hybridize to the target sequence at equilibrium.Typically, stringent conditions will be those in which the saltconcentration is at least about 0.01 to 1.0 M sodium ion concentration(or other salts) at pH 7.0 to 8.3 and the temperature is at least about30° C. for short polynucleotides (e.g., 10 to 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

As used herein, “modulation” of an activity means either an increase(stimulation) or a decrease (inhibition) in that activity. For example,and without limitation, a modulation of proteolysis can mean either anincrease in proteolysis or a decrease in proteolysis.

Latent TGFβ Binding Protein 4 (Ltbp4)

The present disclosure is directed in part to Ltbp4, the gene encodinglatent TGFβ binding protein (LTBP4; GenBank Accession NumberNP_001036009.1; SEQ ID NO: 1), which was identified in a genetic screenas a major genetic modifier of muscular dystrophy [Heydemann et al., JClin Invest. 119: 3703-12 (2009)]. This genetic screen was conductedusing mice lacking the dystrophin-associated protein, γ-sarcoglycan(Sgcg null mice). The Sgcg model of limb girdle muscular dystrophy(LGMD) was selected because there was ample evidence from human LGMD ofthe importance of genetic modifiers affecting the severity of thisdisease [McNally et al., Am J Hum Genet. 59:1040-7 (1996)]. It wassurprisingly found that modifiers identified for sarcoglycan-mediatedmuscular dystrophy similarly modify DMD. Disruption of the dystrophinglycoprotein complex, either in DMD or the sarcoglycan-associated LGMDs,leads to a fragile muscle membrane, enhanced myofiber breakdown, andreplacement of normal muscle tissue by fibrosis. Early in pathology,fibrotic replacement is minimal, but in the advanced DMD patient, themuscle is nearly completely replaced by fibrosis.

LTBP4 is located on human chromosome 19q13.1-q13.2, and is anextracellular matrix protein that binds and sequesters TGFβ (FIG. 1).LTBP4 modifies murine muscular dystrophy through a polymorphism in theLtbp4 gene. There are two common variants of the Ltbp4 gene in mice.Most strains of mice, including the mdx mouse, have the Ltbp4 insertionallele (Ltbp4^(I/I)). Insertion of 36 base pairs (12 amino acids) intothe proline-rich region of LTBP4 encoded by Ltbp4^(I/I) leads to milderdisease. Deletion of 36 bp/12aa in the proline-rich region is associatedwith more severe disease (Ltbp4^(D/D)) (FIG. 2). It was found that theLtbp4 genotype correlated strongly with two different aspects ofmuscular dystrophy pathology, i.e., membrane leakage and fibrosis, andthese features define DMD pathology.

To assess muscle membrane leakage, Evans blue dye (EBD), which cancomplex with serum albumin, and thus is a measure of membranepermeability, was used. EBD is injected intraperitoneally and musclesfrom the injected animals are harvested approximately 8-40 hours later.Muscle membrane leakage was assessed by determining the amount of EBD inmultiple different muscle groups, including quadriceps and otherskeletal muscles. Hydroxyproline content was measured to quantifyfibrosis, and this assay was also performed on multiple different musclegroups. The Ltbp4 genotype was found to account for nearly 40% of thevariance in membrane leakage in quadriceps muscle [Swaggart et al.,Physiol Genomics 43: 24-31 (2011)]. Similarly, the Ltbp4 genotype alsohighly correlated with fibrosis in limb-based skeletal muscles where italso accounted for a significant amount of the variance. Ltbp4 is anunusually strong genetic modifier and acts both on membrane fragility aswell as fibrosis. Accordingly, the present disclosure identifies LTBP4as a target for therapy because it will stabilize the plasma membrane inaddition to reducing fibrosis in patients in need thereof.

As discussed hereinabove, LTBP4 is a matrix-associated protein thatbinds and sequesters TGFβ. TGFβ in this form is the large latentcomplex, which requires further proteolysis to become fully active. Itis expected that matrix-bound latent TGFβ is the least active form withregard to receptor engagement, and therefore represents an ideal step atwhich to inhibit TGFβ release. LTBP4, the fourth member of the LTBPcarrier protein family, is highly expressed in heart, muscle, lung andcolon [Saharinen et al., J Biol Chem. 273: 18459-69 (1998)]. LTBP4protein, like other members of this family, can be proteolyzed withplasmin, which results in TGFβ release [Saharinen et al., J Biol Chem.273: 18459-69 (1998); Ge et al., J Cell Biol. 175: 111-20 (2006)]. The12-amino-acid insertion/deletion alters the susceptibility of LTBP4 toproteolysis, which in turn alters TGFβ release and its ability to bindTGFβ receptors and activate signaling. It is disclosed herein thatinhibiting LTBP4 cleavage will hold TGFβ inactive and limit thedownstream effects of TGFβ release.

Agents

Methods of the disclosure contemplate treating a patient having aTGFβ-related disease comprising administering to the patient aneffective amount of an agent that modulates proteolysis of LTBP4.

The term “agent” in this context refers to an antibody, an inhibitorynucleic acid, a peptide, and combinations thereof.

Antibodies

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)₂, Fv, single chainantibodies, diabodies), camel bodies and recombinant peptides comprisingthe foregoing provided they exhibit the desired biological activity.Antibody fragments may be produced using recombinant DNA techniques orby enzymatic or chemical cleavage of intact antibodies and are describedfurther below. Non-limiting examples of monoclonal antibodies includemurine, chimeric, humanized, human, and human-engineeredimmunoglobulins, antibodies, chimeric fusion proteins having sequencesderived from immunoglobulins, or muteins or derivatives thereof, eachdescribed further below. Multimers or aggregates of intact moleculesand/or fragments, including chemically derivatized antibodies, arecontemplated. Antibodies of any isotype class or subclass arecontemplated.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to conventional(polyclonal) antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, monoclonal antibodies areadvantageous in that they are synthesized in a homogeneous culture,uncontaminated by other immunoglobulins with different specificities andcharacteristics.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the disclosure may be made bythe hybridoma method first described by Kohler et al., Nature 256: 495(1975), or may be made by recombinant DNA methods (see, e.g., U U.S.Pat. No. 4,816,567, incorporated herein by reference). The “monoclonalantibodies” may also be recombinant, chimeric, humanized, human, HumanEngineered™, or antibody fragments, for example.

Antibodies described herein are discussed in Example 3. In certainembodiments, a variant of an antibody of the disclosure is contemplated.By “variant” is meant an antibody comprising one or more amino acidsubstitutions, amino acid deletions, or amino acid additions to areference amino acid sequence. Variants include, but are not limited to,antibodies having an amino acid sequence that is at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to any of the amino acid sequences of an antibody providedherein, provided that the antibody variant retains the ability to blockand/or inhibit the proteolysis of LTBP4.

In further embodiments, an anti-LTBP4 antibody described hereinspecifically binds at least one peptide selected from the groupconsisting of peptides having a sequence set forth in SEQ ID NOs: 2-5,or a peptide selected from the group consisting of peptides having asequence at least 70% identical to a peptide having a sequence set forthin SEQ ID NOs: 2-5. In additional embodiments, an anti-LTBP4 antibodydescribed herein binds at least one epitope of LTBP4 with an affinity of10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less(lower meaning higher binding affinity), or optionally binds all ofLTBP4 with an affinity of 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M 10⁻¹⁰ M, 10⁻¹¹M, or 10⁻¹² M or less. In other embodiments, an antibody describedherein “specifically binds” to LTBP4 with at least 2-50 fold, 10-100fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%,50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higheraffinity compared to binding to a non-target protein.

Antibodies described hereinbelow are suitable for use in the methodsdescribed herein. Additional antibodies are also contemplated, providedthe antibody possesses the property of modulating the proteolysis orupregulating the activity of LTBP4. Such antibodies may, for example, behumanized according to known techniques and modified and/or formulatedto allow delivery and intracellular contact with LTBP4.

Peptides

The disclosure provides peptides that have the ability to act as asubstrate for a protease (i.e., “a protease-substrate peptide”). Theprotease, as discussed herein, means a protease that can cleave LTBP4.In one embodiment, the protease is a serine protease. In furtherembodiments, the protease is selected from the group consisting ofplasmin, leukocyte elastase, pancreatic elastase, human mast cellchymase, trypsin, chymotrypsin, pepsin and papain.

The ability of a peptide to act as a substrate for a protease can bereadily determined by one of ordinary skill in the art. By way ofnon-limiting example, a peptide can be tested in vitro by incubating alabeled LTBP4 protein (or a labeled fragment thereof) with a candidatepeptide and a serine protease. The label can be any detectable labelknown in the art, and in one embodiment is a radioactive label.Following incubation and subsequent gel electrophoresis, it can bedetermined whether the LTBP4 protein (or fragment thereof) wasrefractory to proteolysis based on the size of the protein on the gel.If the LTBP4 protein was not protected from proteolysis by the peptide,the radioactive band on the gel will be smaller than one would expectfor a full-length LTBP4 protein. Thus, while peptide sequences disclosedherein are contemplated for use according to the methods of thedisclosure, additional peptides are also contemplated, with the provisobeing their ability to act as a substrate for a protease in a mannerthat renders them an inhibitor of LTBP4 proteolysis.

Use of one or more peptides or antibodies of the disclosure, each ofwhich has an ability to act either as a substrate for a protease(peptide) or to act as an inhibitor of proteolysis (antibody), isexpected to upregulate the activity of LTBP4 compared to the activity ofLTBP4 in the absence of the one or more peptides. In this context,upregulation of LTBP4 activity results from its protection fromproteolysis via the action of the one or more peptides and/or antibodiesof the disclosure. The downstream effect of this upregulation of LTBP4activity is the concomitant downregulation of TGFβ signaling. IntactLTBP4 will continue to bind and sequester TGFβ and thus prevent itsrelease and subsequent downstream effects. Thus, in various embodiments,the upregulation of LTBP4 activity is measured by quantitating TGFβsignaling. Methods of quantitating TGFβ signaling are known to those ofskill in the art, and include determination of Smad signaling from abiological sample obtained from a patient. It is contemplated that, insome embodiments, Smad signaling in a patient being administered one ormore agent(s) and/or additional agent(s) of the disclosure is reduced byat least about 1% and up to about 5%, about 10%, about 20%, about 30%,about 40% or about 50% relative to a patient not so treated. In furtherembodiments, Smad signaling in a patient being administered one or moreagent(s) and/or additional agent(s) of the disclosure is reduced by atleast about 10% and up to about 20%, about 50%, about 70%, about 80%,about 90%, about 99% or more relative to a patient not so treated. Inspecific embodiments, Smad signaling in a patient being administered oneor more agent(s) and/or additional agent(s) of the disclosure is reducedby at least about 1%, about 2%, about 5%, about 10%, about 20%, about25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 99% or more relative to a patientnot so treated.

Peptides described above (i.e., peptide inhibitors of LTBP4 proteolysis)are set forth in Example 3 and Table 1. Thus, in certain embodiments,the peptide comprises or consists of the amino acid sequence of any oneof SEQ ID NOs: 2-5 or a variant of any of the foregoing. By “variant” ismeant a peptide comprising one or more amino acid substitutions, aminoacid deletions, or amino acid additions to a reference amino acidsequence (e.g., any one of SEQ ID NOs: 2-5). Variants include, but arenot limited to, peptides having an amino acid sequence that is at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to any of the amino acid sequences provided hereinwhile retaining the ability to act as a substrate for a protease.

In one aspect, the peptide consists of 35 amino acids or less. Invarious embodiments, the peptide comprises 15-35 amino acid residues(e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, or 35 amino acid residues). It is also contemplated thata peptide described herein comprising one or more deletions is suitablein the context of the disclosure so long as the peptide can act as asubstrate for a protease. In some embodiments, amino acids are removedfrom within the amino acid sequence, at the N-terminus, and/or at theC-terminus. Such peptide fragments can comprise 3-14 amino acid residues(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues).

Optionally, the peptide comprises one or more amino acid substitutions(with reference to any of the amino acid sequences provided herein) thatdo not destroy the ability of the peptide to act as a substrate for aprotease. Amino acid substitutions include, but are not limited to,those which: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinities, and/or (4)confer or modify other physiochemical or functional properties on apeptide. In one aspect, the substitution is a conservative substitution,wherein an amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined within the art, and include aminoacids with basic side chains (e.g., lysine, arginine, and histidine),acidic side chains (e.g., aspartic acid and glutamic acid), unchargedpolar side chains (e.g., glycine, asparagine, glutamine, serine,threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan), beta-branched side chains (e.g., threonine, valine, andisoleucine) and side chains with aromatic character (e.g., tyrosine,phenylalanine, tryptophan, and histidine). It will be appreciated,however, that a practitioner is not limited to conservativesubstitutions, so long as the resulting peptide retains the ability toact as a substrate, in whole or in part, for a protease. The disclosurealso embraces protease-substrate peptides comprising atypical,non-naturally occurring amino acids, which are well known in the art.The individual amino acids may have either L or D stereochemistry whenappropriate, although the L stereochemistry is typically employed forall of the amino acids in the peptide.

The disclosure further includes protease-substrate peptide variantscomprising one or more amino acids inserted within an amino acidsequence provided herein and/or attached to the N-terminus orC-terminus. In some embodiments, the peptide further comprises one ormore amino acids that facilitate synthesis, handling, or use of thepeptide including, but not limited to, one or two lysines at theN-terminus and/or C-terminus to increase solubility of the peptide.Suitable fusion proteins include, but are not limited to, proteinscomprising a peptide linked to another polypeptide, a polypeptidefragment, or amino acids not generally recognized to be part of theprotein sequence. In some embodiments, a fusion peptide comprises theentire amino acid sequences of two or more peptides or, alternatively,comprises portions (fragments) of two or more peptides. In addition toall or part of the peptides described herein, a fusion proteinoptionally includes all or part of any suitable peptide comprising adesired biological activity/function. Indeed, in some embodiments, apeptide is operably linked to, for instance, one or more of thefollowing: a peptide with long circulating half-life, a marker protein,a peptide that facilitates purification of the protease-substratepeptide, a peptide sequence that promotes formation of multimericproteins, or a fragment of any of the foregoing. In one embodiment, twoor more protease-substrate peptides are fused together, linked by amultimerization domain, or attached via chemical linkage to generate aprotease-substrate peptide complex. The protease-substrate peptides maybe the same or different.

“Derivatives” are also contemplated by the disclosure and includeprotease-substrate peptides that have been chemically modified in somemanner distinct from addition, deletion, or substitution of amino acids.In this regard, a peptide provided herein is chemically bonded withpolymers, lipids, other organic moieties, and/or inorganic moieties.Derivatives are prepared in some situations to increase solubility,absorption, or circulating half-life. Various chemical modificationseliminate or attenuate any undesirable side effect of the agent. In thisregard, the disclosure provides protease-substrate peptides covalentlymodified to include one or more water-soluble polymer attachments.Useful polymers known in the art include, but are not limited to,polyethylene glycol, polyoxyethylene glycol, polypropylene glycol,monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of any ofthe foregoing. For further discussion of water soluble polymerattachments, see U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; and 4,179,337, incorporated herein by reference.In other embodiments, a peptide derivative includes a targeting moietyspecific for a particular cell type, tissue, and/or organ.Alternatively, the peptide is linked to one or more chemical moietiesthat facilitate purification, detection, multimerization, and/orcharacterization of peptide activity.

Derivatives also include peptides comprising modified ornon-proteinogenic amino acids or a modified linker group [see, e.g.,Grant, Synthetic Peptides: A User's Guide, Oxford University Press(1992)]. Modified amino acids include, for example, amino acids whereinthe amino and/or carboxyl group is replaced by another group.Non-limiting examples include modified amino acids incorporatingthioamides, ureas, thioureas, acylhydrazides, esters, olefines,sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acidamides, benzodiazepines and other aromatic or non-aromatic heterocycles[see Estiarte et al., Burgers Medicinal Chemistry, 6th edition, Volume1, Part 4, John Wiley & Sons, New York (2002)]. Modified amino acids areoften connected to the peptide with at least one of the above-mentionedfunctional groups instead of an amide bond. Non-proteinogenic aminoacids include, but are not limited, to β-alanine (β-Ala), norvaline(Nva), norleucine (Nle), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyricacid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn),hydroxyproline (Hyp), sarcosine, citrulline, cysteic acid (Coh),cyclohexylalanine, methioninesulfoxide (Meo), methioninesulfone (Moo),homoserinemethylester (Hsm), propargylglycine (Eag), 5-fluorotryptophan(5Fw), 6-fluorotryptophan (6Fw), 3′,4′-dimethoxyphenyl-alanine (Ear),3′,4′-difluorophenylalanine (Dff), 4′-fluorophenyl-alanine (Pff),1-naphthyl-alanine (1Ni), 1-methyltryptophan (1Mw), penicillamine (Pen),homoserine (HSe), α-amino isobutyric acid, t-butylglycine,t-butylalanine, phenylglycine (Phg), benzothienylalanine (Bta),L-homo-cysteine (L-Hcys), N-methyl-phenylalanine (NMF), 2-thienylalanine(Thi), 3,3-diphenylalanine (Ebw), homophenylalanine (Hfe),s-benzyl-L-cysteine (Ece) and cyclohexylalanine (Cha). These and othernon-proteinogenic amino acids may exist as D- or L-isomers and D-isomersof proteinogenic amino acids may also be found in derivatives.

Examples of modified linkers include, but are not limited to, theflexible linker 4,7,10-trioxa-1,13-tridecanediamine (Ttds), glycine,6-aminohexanoic acid, beta-alanine, and combinations of Ttds, glycine,6-aminohexanoic acid and beta-alanine.

Protease-substrate peptides are made in a variety of ways. In someembodiments, the peptides are synthesized by solid-phase synthesistechniques including those described in Merrifield, J. Am. Chem. Soc.85: 2149 (1963); Davis et al., Biochem. Intl. 10: 394-414 (1985); Larsenet al., J. Am. Chem. Soc. 115: 6247 (1993); Smith et al., J. PeptideProtein Res. 44:183 (1994); O'Donnell et al., J. Am. Chem. Soc. 118:6070 (1996); Stewart and Young, Solid Phase Peptide Synthesis, Freeman(1969); Finn et al., The Proteins, 3rd ed., vol. 2, pp. 105-253 (1976);and Erickson et al., The Proteins, 3rd ed., vol. 2, pp. 257-527 (1976).Alternatively, the protease-substrate peptide is expressed recombinantlyby introducing a nucleic acid encoding a protease-substrate peptide intohost cells that are cultured to express the peptide. Such peptides arepurified from the cell culture using standard protein purificationtechniques.

The disclosure also encompasses a nucleic acid comprising a nucleic acidsequence encoding an antibody or protease-substrate peptide. Methods ofpreparing DNA and/or RNA molecules are well known in the art. In oneaspect, a DNA/RNA molecule encoding an antibody or protease-substratepeptide provided herein is generated using chemical synthesis techniquesand/or using polymerase chain reaction (PCR). If desired, an antibodyand/or protease-substrate peptide coding sequence is incorporated intoan expression vector. One of ordinary skill in the art will appreciatethat any of a number of expression vectors known in the art are suitablein the context of the disclosure, such as, but not limited to, plasmids,plasmid-liposome complexes, and viral vectors. Any of these expressionvectors are prepared using standard recombinant DNA techniques describedin, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2dedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y. (1994).Optionally, the nucleic acid is operably linked to one or moreregulatory sequences, such as a promoter, activator, enhancer, capsignal, polyadenylation signal, or other signal involved in the controlof transcription or translation.

As with all binding agents and binding assays, one of skill in this artrecognizes that the various moieties to which a binding agent should notdetectably bind in order to be biologically (e.g., therapeutically)effective would be exhaustive and impractical to list. Therefore, whendiscussing a peptide, the term “specifically binds” refers to theability of a peptide to bind (or otherwise inhibit) a protease involvedin cleavage of LTBP4 with greater affinity than it binds to a non-targetcontrol protein that is not the protease. For example, the peptide maybind to the protease with an affinity that is at least, 5, 10, 15, 25,50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for acontrol protein. In some embodiments, the peptide binds the proteasewith greater affinity than it binds to an “anti-target,” a protein orother naturally occurring substance in humans wherein binding of thepeptide might lead to adverse effects. Several classes of peptides arepotential anti-targets. Because protease-substrate peptides are expectedto exert their activity in the extracellular matrix, ECM proteins arecontemplated as anti-targets.

Also specifically contemplated by the disclosure are peptides thatelicit an immune response to LTBP4 in methods to modulate LTBP4 thatinvolve the host immune system. Thus, in some aspects, a composition isprovided that comprises a peptide of the disclosure for use as a vaccinein an individual. Vaccines often include an adjuvant. The compositionscomprising one or more peptides described herein may also contain anadjuvant, or be administered with an adjuvant. Thus, the adjuvant may beadministered with the peptide compositions or as part of the peptidecompositions, before the peptide compositions, or after the peptidecompositions.

A variety of adjuvants are suitable for use in combination with thepeptide composition to elicit an immune response to the peptide.Preferred adjuvants augment the intrinsic response to an antigen withoutcausing conformational changes in the antigen that affect thequalitative form of the response. Adjuvants for use in the methodsdisclosed herein include, but are not limited to, keyhole limpethemocyanin (KLH), forms of alum (see below) and 3 De-O-acylatedmonophosphoryl lipid A (MPL or 3-DMP) [see GB 2220211]. Other suitableadjuvant include QS21, which is a triterpene glycoside or saponinisolated from the bark of the Quillaja Saponaria Molina tree found inSouth America [see Kensil et al., in Vaccine Design: The Subunit andAdjuvant Approach (eds. Powell and Newman, Plenum Press, N Y, 1995);U.S. Pat. No. 5,057,540] and CpG [Bioworld Today, Nov. 15, 1998]. Stillother suitable adjuvants are described in the following paragraph.

One class of suitable adjuvants, noted briefly above, is aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, and aluminumsulfate. Such adjuvants can be used with or without other specificimmunostimulating agents such as MPL or 3-DMP, QS21, polymeric ormonomeric amino acids such as polyglutamic acid or polylysine. Anotherclass of suitable adjuvants is oil-in-water emulsion formulations [suchas squalene or peanut oil], optionally in combination with immunologicalstimulants, such as monophosphoryl lipid A [see Stoute et al., N. Engl.J. Med. 336, 86-91 (1997)]. Such adjuvants can be used with or withoutother specific immunostimulating agents such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))ethylamide (MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wallcomponents. Additional oil-in-water emulsions include (a) MF59 (WO90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE) formulated intosubmicron particles using a microfluidizer such as a Model 110Ymicrofluidizer (Microfluidics, Newton Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP,either microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) the Ribi adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another classof suitable adjuvants is saponin adjuvants, such as Stimulon™ (QS21,Aquila, Worcester, Mass.) or particles generated therefrom such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA). Any of the suitable adjuvants may include a cytokine,such as an interleukin (IL-1, IL-2, or IL-12), macrophage colonystimulating factor (M-CSF), tumor necrosis factor (TNF), or combinationsof cytokines.

An adjuvant can be administered with a peptide composition of thedisclosure as a single composition, or can be administered before,concurrent with or after administration of a peptide composition of thedisclosure. Immunogen and adjuvant can be packaged and supplied in thesame vial or can be packaged in separate vials and mixed before use.Immunogen and adjuvant are typically packaged with a label indicatingthe intended application, such as a therapeutic application. Ifimmunogen and adjuvant are packaged separately, the packaging typicallyincludes instructions for mixing before use. The choice of an adjuvantand/or carrier depends on the stability of the vaccine containing theadjuvant, the route of administration, the dosing schedule, the efficacyof the adjuvant for the species being vaccinated, and, in humans, apharmaceutically acceptable adjuvant is one that has been approved or isapprovable for human administration by pertinent regulatory agencies.For example, Complete Freund's adjuvant is not suitable for humanadministration, while alum, MPL and QS21 are suitable. Optionally, twoor more different adjuvants can be used simultaneously, such as alumwith MPL, alum with QS21, MPL with QS21, and alum, QS21 and MPLtogether. Also, Incomplete Freund's adjuvant can be used [Chang et al.,Advanced Drug Delivery Reviews 32, 173-186 (1998)], optionally incombination with any of alum, QS21, and MPL and all combinationsthereof.

Inhibitory Nucleic Acids

By “inhibitory nucleic acid” is meant an RNA or DNA polynucleotide thatbinds to another RNA or DNA (target RNA, DNA). An inhibitory nucleicacid downregulates expression and/or function of a particular targetpolynucleotide. The definition is meant to include any foreign RNA orDNA molecule which is useful from a therapeutic, diagnostic, or otherviewpoint. Such molecules include, for example, antisensepolynucleotides such as RNA or DNA molecules, interference RNA (RNAi),micro RNA (miRNA), siRNA, enzymatic RNA, aptamers, ribozymes and otherpolymeric compounds that hybridize to at least a portion of the targetpolynucleotide or target polypeptide. As such, these compounds may beintroduced in the form of single-stranded, double-stranded,triple-stranded, or partially single-stranded molecules, and themolecules may be linear or circular polymeric compounds.

The production and use of aptamers is known to those of ordinary skillin the art. In general, aptamers are nucleic acid- or peptide-bindingspecies capable of tightly binding to and discreetly distinguishingtarget ligands [Yan et al., RNA Biol. 6(3): 316-320 (2009), incorporatedby reference herein in its entirety]. Aptamers, in some embodiments, maybe obtained by a technique called the systematic evolution of ligands byexponential enrichment (SELEX) process [Tuerk et al., Science 249:505-10 (1990), U.S. Pat. No. 5,270,163, and U.S. Pat. No. 5,637,459,each of which is incorporated herein by reference in its entirety].General discussions of nucleic acid aptamers are found in, for exampleand without limitation, Nucleic Acid and Peptide Aptamers: Methods andProtocols (Edited by Mayer, Humana Press, 2009) and Crawford et al.,Briefings in Functional Genomics and Proteomics 2(1): 72-79 (2003). Invarious aspects, an aptamer is between 10-100 nucleotides in length.

As used herein, the term “target polynucleotide” encompasses DNA, RNA(comprising pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA, coding sequences, noncoding sequences, sensepolynucleotides or antisense polynucleotides. The specific hybridizationof a polymeric compound with its target nucleic acid interferes with thenormal function of the target nucleic acid. This modulation of functionof a target nucleic acid or polynucleotide by compounds thatspecifically hybridize to it is generally referred to as antisensemodulation or inhibition. The functions of DNA to be interfered include,for example, replication and transcription. The functions of RNA to beinterfered include all vital functions such as, for example and withoutlimitation, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, catalytic activity which may be engaged in orfacilitated by the RNA, and/or translation to express an encodedpolypeptide. The overall effect of such modulation (e.g., inhibition)with target polynucleotide function is modulation of the expression ofan encoded product or activity of the polynucleotide itself.

RNA interference “RNAi” is mediated by double-stranded RNA (dsRNA)molecules that have sequence-specific homology to their target nucleicacid sequence(s) [Caplen et al., Proc. Natl. Acad Sci. USA 98: 9742-9747(2001)]. In certain embodiments of the present disclosure, the mediatorsare “small interfering” RNA duplexes (siRNAs) of 5-25 nucleotides. ThesiRNAs are derived from the processing of dsRNA by an RNase enzyme knownas Dicer [Bernstein et al., Nature 409: 363-366 (2001)]. The siRNAduplex products are recruited into a multi-protein siRNA complex termedRISC (RNA-Induced Silencing Complex). Small interfering RNAs that can beused in accordance with the present disclosure can be synthesized andused according to procedures that are well known in the art and thatwill be familiar to the ordinarily skilled artisan. The siRNAs for usein the methods of the present disclosure suitably comprise between about1 to about 50 nucleotides (nt). In non-limiting embodiments, siRNAscomprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 toabout 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.

Methods for inhibiting target polynucleotide expression are providedthat include those wherein expression of the target polynucleotide isinhibited by at least about 5% and up to about 10%, 20%, 50% or 100%, atleast about 5% and up to about 30%, 60%, 70% or 90%, or at least 10% andup to about 50%, 60%, 70%, 80%, 90% or 100%. In additional embodiments,expression of the target polynucleotide is inhibited by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or 100% compared to target polynucleotide expressionin the absence of an inhibitory nucleic acid. In other words, methods ofinhibiting the expression or activity of a polynucleotide according tothe disclosure result in essentially any degree of inhibition ofexpression of a target polynucleotide.

The degree of inhibition is determined in vivo from a body fluid sampleor from a biopsy sample or by imaging techniques well known in the art.Alternatively, the degree of inhibition is determined in a cell cultureassay, generally as a predictable measure of a degree of inhibition thatcan be expected in vivo resulting from use of a specific type ofspecific inhibitory nucleic acid.

Exon Skipping

Inhibitory nucleic acids are also contemplated for use in exon skipping.In general, exon skipping is a method in which inhibitory nucleic acidsare designed to modulate the splicing of a gene of interest, resultingin mRNA transcripts that are able to make some level of gene productwith some function. The inhibitory nucleic acids are, in variousembodiments, short nucleic acid sequences designed to selectively bindto specific mRNA or pre-mRNA sequences to generate small double-strandedregions of the target mRNA. By binding to these regions and formingdouble strands at key sites where the spliceosome or proteins of thespliceosome would normally bind, mutated (frameshifting) exons areskipped and the remainder of the pre-mRNA is edited correctly in frame,albeit shorter.

Exon skipping is generally described in Hoffman et al. [The American J.of Path. 179(1): 12-22 (2010], Lu et al. [The American Soc. of Gene andCell Therapy 19(1): 9-15 (2011)], and U.S. Pat. Nos. 8,084,601,7,960,541 and 7,973,015, all of which are incorporated herein byreference in their entireties.

Thus, the disclosure contemplates that skipping exons that encode theproline-rich region of LTBP4 will generate a protease-resistant protein.In some embodiments, one or more of exons 11, 12 and 13 of mouse LTBP4(corresponding to exons 11, 12 and 13 in human LTBP4) are targeted forexon skipping. It is expected that skipping of the exons that encodepart or all of the proline-rich region of LTBP4 will generate a proteinthat is resistant to protease activity.

Compositions

Any of the agents and/or additional agents described herein (or nucleicacids encoding any of the agents and/or additional agents describedherein) also is provided in a composition. In this regard, the agentand/or additional agent is formulated with a physiologically-acceptable(i.e., pharmacologically acceptable) carrier, buffer, or diluent, asdescribed further herein. Optionally, the peptide is in the form of aphysiologically acceptable salt, which is encompassed by the disclosure.“Physiologically acceptable salts” means any salts that arepharmaceutically acceptable. Some examples of appropriate salts includeacetate, trifluoroacetate, hydrochloride, hydrobromide, sulfate,citrate, tartrate, glycolate, and oxalate.

TGFβ-Related Diseases

TGFβ-related diseases contemplated for treatment according to thedisclosure include Duchenne Muscular Dystrophy, Limb Girdle MuscularDystrophy, Becker Muscular Dystrophy, myopathy, cystic fibrosis,pulmonary fibrosis, cardiomyopathy, acute lung injury, acute muscleinjury, acute myocardial injury, radiation-induced injury, colon cancer,idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia,autoimmune lung diseases, benign prostate hypertrophy, cerebralinfarction, musculoskeletal fibrosis, post-surgical adhesions, livercirrhosis, renal fibrotic disease, fibrotic vascular disease,neurofibromatosis, Alzheimer's disease, diabetic retinopathy, skinlesions, lymph node fibrosis associated with HIV, chronic obstructivepulmonary disease (COPD), inflammatory pulmonary fibrosis, rheumatoidarthritis; rheumatoid spondylitis; osteoarthritis; gout, other arthriticconditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis;toxic shock syndrome; myofacial pain syndrome (MPS); Shigellosis;asthma; adult respiratory distress syndrome; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; glomerularnephritis; scleroderma; chronic thyroiditis; Grave's disease; Ormond'sdisease; autoimmune gastritis; myasthenia gravis; autoimmune hemolyticanemia; autoimmune neutropenia; thrombocytopenia; pancreatic fibrosis;chronic active hepatitis including hepatic fibrosis; renal fibrosis,irritable bowel syndrome; pyresis; restenosis; cerebral malaria; strokeand ischemic injury; neural trauma; Huntington's disease; Parkinson'sdisease; allergies, including allergic rhinitis and allergicconjunctivitis; cachexia; Reiter's syndrome; acute synoviitis; muscledegeneration, bursitis; tendonitis; tenosynoviitis; osteopetrosis;thrombosis; silicosis; pulmonary sarcosis; bone resorption diseases,such as osteoporosis or multiple myeloma-related bone disorders; cancer,including but not limited to metastatic breast carcinoma, colorectalcarcinoma, malignant melanoma, gastric cancer, and non-small cell lungcancer; graft-versus-host reaction; and auto-immune diseases, such asmultiple sclerosis, lupus and fibromyalgia; viral diseases such asHerpes Zoster, Herpes Simplex I or II, influenza virus, Severe AcuteRespiratory Syndrome (SARS) and cytomegalovirus.

As used herein, “cardiomyopathy” refers to any disease or dysfunction ofthe myocardium (heart muscle) in which the heart is abnormally enlarged,thickened and/or stiffened. As a result, the heart muscle's ability topump blood is usually weakened, often leading to congestive heartfailure. The disease or disorder can be, for example, inflammatory,metabolic, toxic, infiltrative, fibrotic, hematological, genetic, orunknown in origin. Such cardiomyopathies may result from a lack ofoxygen. Other diseases include those that result from myocardial injurywhich involves damage to the muscle or the myocardium in the wall of theheart as a result of disease or trauma. Myocardial injury can beattributed to many things such as, but not limited to, cardiomyopathy,myocardial infarction, or congenital heart disease. The cardiac disordermay be pediatric in origin. Cardiomyopathy includes, but is not limitedto, cardiomyopathy (dilated, hypertrophic, restrictive, arrhythmogenic,genetic, idiopathic and unclassified cardiomyopathy), sporadic dilatedcardiomyopathy, X-linked Dilated Cardiomyopathy (XLDC), acute andchronic heart failure, right heart failure, left heart failure,biventricular heart failure, congenital heart defects, myocardiacfibrosis, mitral valve stenosis, mitral valve insufficiency, aorticvalve stenosis, aortic valve insufficiency, tricuspidal valve stenosis,tricuspidal valve insufficiency, pulmonal valve stenosis, pulmonal valveinsufficiency, combined valve defects, myocarditis, acute myocarditis,chronic myocarditis, viral myocarditis, diastolic heart failure,systolic heart failure, diabetic heart failure and accumulationdiseases.

TGFβ Proteins

The disclosure provides compositions and methods directed to modulatingthe activity, including the expression, of LTBP4, which is a proteinthat interacts with TGFβ proteins. Modulation of the activity of anyprotein that interacts with LTBP4 is contemplated by the disclosure, andin various embodiments the TGFβ protein is selected from the groupconsisting of a growth and differentiation factor (GDF), activin,inhibin, and a bone morphogenetic protein. TGFβ proteins are known inthe art and are discussed, for example and without limitation, inSchmierer et al. (Nature Reviews Molecular Cell Biology 8: 970-982(2007)], incorporated herein by reference. In addition, isoforms of TGFβproteins are contemplated and include, without limitation, TGFβ 1, TGFβ2, TGFβ 3, GDF 8, and GDF 11.

Practice of methods of the disclosure wherein a patient is administeredone or more agent(s) and optionally additional agent(s) is expected toresult in modulation of the activity of a TGFβ protein by at least about1% relative to a patient not so treated. In further embodiments, theactivity of a TGFβ protein in a patient that is administered one or moreagent(s) and/or additional agent(s) is modulated by at least about 1%and up to any one of about 2%, about 5%, about 10% or about 15% TGFβactivity relative to a patient not so treated. In still furtherembodiments, the activity of a TGFβ protein in a patient that isadministered one or more agent(s) and optionally additional agent(s) ismodulated by at least about 10% and up to any one of about 15%, about20%, about 25% or about 30% TGFβ activity relative to a patient not sotreated. In further embodiments, the activity of a TGFβ protein in apatient that is administered one or more agent(s) and/or additionalagent(s) is modulated by at least about 10% and up to any one of about40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,about 99% or more TGFβ activity relative to a patient not so treated. Inspecific embodiments, the activity of a TGFβ protein in a patient thatis administered one or more agent(s) and optionally additional agent(s)is modulated by at least about 1%, about 2%, about 5%, about 10%, about20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 99% TGFβ activity ormore relative to a patient not so treated. Protein activity may bequantitated by methods generally known to those of skill in the art.

Additional (Second) Agents

In various embodiments of the disclosure it is contemplated that asecond agent is administered with the agent that modulates LTBP4activity by modulating the proteolysis of LTBP4. Nonlimiting examples ofthe second agent are a modulator of an inflammatory response, a promoterof muscle growth, a chemotherapeutic agent and a modulator of fibrosis.Further, the methods disclosed herein can, in various embodiments,encompass one or more of such agents, or one or more of such agents incomposition with any other active agent(s).

Chemotherapeutic Agents

Chemotherapeutic agents contemplated for use include, withoutlimitation, alkylating agents including: nitrogen mustards, such asmechlor-ethamine, cyclophosphamide, ifosfamide, melphalan andchlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU),and semustine (methyl-CCNU); ethylenimines/methylmelamine such asthriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),hexamethylmelamine (HMM, altretamine); alkyl sulfonates such asbusulfan; triazines such as dacarbazine (DTIC); antimetabolitesincluding folic acid analogs such as methotrexate and trimetrexate,pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine,gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine,2,2′-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine,6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin),erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products includingantimitotic drugs such as paclitaxel, vinca alkaloids includingvinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine,and estramustine phosphate; epipodophylotoxins such as etoposide andteniposide; antibiotics such as actimomycin D, daunomycin (rubidomycin),doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin(mithramycin), mitomycin C, and actinomycin; enzymes such asL-asparaginase; biological response modifiers such as interferon-alpha,IL-2, G-CSF and GM-CSF; miscellaneous agents including platinumcoordination complexes such as cisplatin and carboplatin,anthracenediones such as mitoxantrone, substituted urea such ashydroxyurea, methylhydrazine derivatives including N-methylhydrazine(MIH) and procarbazine, adrenocortical suppressants such as mitotane(o,p′-DDD) and aminoglutethimide; hormones and antagonists includingadrenocorticosteroid antagonists such as prednisone and equivalents,dexamethasone and aminoglutethimide; progestin such ashydroxyprogesterone caproate, medroxyprogesterone acetate and megestrolacetate; estrogen such as diethylstilbestrol and ethinyl estradiolequivalents; antiestrogen such as tamoxifen; androgens includingtestosterone propionate and fluoxymesterone/equivalents; antiandrogenssuch as flutamide, gonadotropin-releasing hormone analogs andleuprolide; and non-steroidal antiandrogens such as flutamide.

Modulators of Fibrosis

A “modulator of fibrosis” as used herein is synonymous with antifibroticagent. The term “antifibrotic agent” refers to a chemical compound thathas antifibrotic activity (i.e., prevents or reduces fibrosis) inmammals. This takes into account the abnormal formation of fibrousconnective tissue, which is typically comprised of collagen. Thesecompounds may have different mechanisms of action, some reducing theformation of collagen or another protein, others enhancing thecatabolism or removal of collagen in the affected area of the body. Allsuch compounds having activity in the reduction of the presence offibrotic tissue are included herein, without regard to the particularmechanism of action by which each such drug functions. Antifibroticagents useful in the methods and compositions of the disclosure includethose described in U.S. Pat. No. 5,720,950, incorporated herein byreference. Additional antifibrotic agents contemplated by the disclosureinclude, but are not limited to, Type II interferon receptor agonists(e.g., interferon-gamma); pirfenidone and pirfenidone analogs;anti-angiogenic agents, such as VEGF antagonists, VEGF receptorantagonists, bFGF antagonists, bFGF receptor antagonists, TGFβantagonists, TGFβ receptor antagonists; anti-inflammatory agents, IL-1antagonists, such as IL-1Ra, angiotensin-converting-enzyme (ACE)inhibitors, angiotensin receptor blockers and aldosterone antagonists.

Modulators of an Inflammatory Response

A modulator of an inflammatory response includes the following agents.In one embodiment of the disclosure, the modulator of an inflammatoryresponse is a beta2-adrenergic receptor agonist (e.g., albuterol). Theterm beta2-adrenergic receptor agonist is used herein to define a classof drugs which act on the β2-adrenergic receptor, thereby causing smoothmuscle relaxation resulting in dilation of bronchial passages,vasodilation in muscle and liver, relaxation of uterine muscle andrelease of insulin. In one embodiment, the beta2-adrenergic receptoragonist for use according to the disclosure is albuterol, animmunosuppressant drug that is widely used in inhalant form forasthmatics. Albuterol is thought to slow disease progression bysuppressing the infiltration of macrophages and other immune cells thatcontribute to inflammatory tissue loss. Albuterol also appears to havesome anabolic effects and promotes the growth of muscle tissue.Albuterol may also suppress protein degradation (possibly via calpaininhibition).

In DMD, the loss of dystrophin leads to breaks in muscle cell membrane,and destabilizes neuronal nitric oxide synthase (nNOS), a protein thatnormally generates nitric oxide (NO). It is thought that at least partof the muscle degeneration observed in DMD patients may result from thereduced production of muscle membrane-associated neuronal nitric oxidesynthase. This reduction may lead to impaired regulation of thevasoconstrictor response and eventual muscle damage.

In one embodiment, modulators of an inflammatory response suitable foruse in compositions of the disclosure are Nuclear Factor Kappa-B (NF-κB)inhibitors. NF-κB is a major transcription factor modulating cellularimmune, inflammatory and proliferative responses. NF-κB functions inactivated macrophages to promote inflammation and muscle necrosis and inskeletal muscle fibers to limit regeneration through the inhibition ofmuscle progenitor cells. The activation of this factor in DMDcontributes to diseases pathology. Thus, NF-kB plays an important rolein the progression of muscular dystrophy and the IKK/NF-κB signalingpathway is a potential therapeutic target for the treatment of aTGFβ-related disease. Inhibitors of NF-κB (for example, IRFI 042, avitamin E analog) enhance muscle function, decrease serum creatinekinase (CK) level and muscle necrosis and enhance muscle regeneration.Furthermore, specific inhibition of NF-κB-mediated signaling by IKK hassimilar benefits.

In a further embodiment, the modulator of an inflammatory response is atumor necrosis factor alpha antagonist. TNF-α is one of the keycytokines that triggers and sustains the inflammation response. In onespecific embodiment of the disclosure, the modulator of an inflammatoryresponse is the TNF-α antagonist infliximab.

TNF-α antagonists for use according to the disclosure include, inaddition to infliximab (Remicade™), a chimeric monoclonal antibodycomprising murine VK and VH domains and human constant Fc domains. Thedrug blocks the action of TNF-α by binding to it and preventing it fromsignaling the receptors for TNF-α on the surface of cells. Another TNF-αantagonist for use according to the disclosure is adalimumab (Humira™)Adalimumab is a fully human monoclonal antibody. Another TNF-αantagonist for use according to the disclosure is etanercept (Enbrel™).Etanercept is a dimeric fusion protein comprising soluble human TNFreceptor linked to an Fc portion of an IgG1. It is a large molecule thatbinds to TNF-α and thereby blocks its action. Etanercept mimics theinhibitory effects of naturally occurring soluble TNF receptors, but asa fusion protein it has a greatly extended half-life in the bloodstreamand therefore a more profound and long-lasting inhibitory effect.

Another TNF-α antagonist for use according to the disclosure ispentoxifylline (Trental™), chemical name1-(5-oxohexyl)-3,7-dimethylxanthine. The usual dosage incontrolled-release tablet form is one tablet (400 mg) three times a daywith meals.

Dosing: Remicade is administered by intravenous infusion, typically at2-month intervals. The recommended dose is 3 mg/kg given as anintravenous infusion followed with additional similar doses at 2 and 6weeks after the first infusion, then every 8 weeks thereafter. Forpatients who have an incomplete response, consideration may be given toadjusting the dose up to 10 mg/kg or treating as often as every 4 weeks.Humira is marketed in both preloaded 0.8 ml (40 mg) syringes and also inpreloaded pen devices, both injected subcutaneously, typically by thepatient at home. Etanercept can be administered at a dose of 25 mg(twice weekly) or 50 mg (once weekly).

In another embodiment of the disclosure, the modulator of aninflammatory response is cyclosporin. Cyclosporin A, the main form ofthe drug, is a cyclic nonribosomal peptide of 11 amino acids produced bythe fungus Tolypocladium inflatum. Cyclosporin is thought to bind to thecytosolic protein cyclophilin (immunophilin) of immunocompetentlymphocytes (especially T-lymphocytes). This complex of cyclosporin andcyclophylin inhibits calcineurin, which under normal circumstances isresponsible for activating the transcription of interleukin-2. It alsoinhibits lymphokine production and interleukin release and thereforeleads to a reduced function of effector T-cells. It does not affectcytostatic activity. It has also an effect on mitochondria, preventingthe mitochondrial PT pore from opening, thus inhibiting cytochrome crelease (a potent apoptotic stimulation factor). Cyclosporin may beadministered at a dose of 1-10 mg/kg/day.

A Promoter of Muscle Growth

In some embodiments of the disclosure, a therapeutically effectiveamount of a promoter of muscle growth is administered to a patient.Promoters of muscle growth contemplated by the disclosure include, butare not limited to, insulin-like growth factor-1 (IGF-1), Akt/proteinkinase B, clenbuterol, creatine, decorin (see U.S. Patent PublicationNumber 20120058955), a steroid (for example and without limitation, acorticosteroid or a glucocorticoid steroid), testosterone and amyostatin antagonist.

Myostatin Antagonist

Another class of promoters of muscle growth suitable for use in thecombinations of the disclosure is myostatin antagonists. Myostatin, alsoknown as growth/differentiation factor 8 (GDF-8) is a transforminggrowth factor-β (TGFβ) superfamily member involved in the regulation ofskeletal muscle mass. Most members of the TGF-β-GDF family are widelyexpressed and are pleiotropic; however, myostatin is primarily expressedin skeletal muscle tissue where it negatively controls skeletal musclegrowth. Myostatin is synthesized as an inactive preproprotein which isactivated by proteolyic cleavage. The precurser protein is cleaved toproduce an approximately 109-amino-acid COOH-terminal protein which, inthe form of a homodimer of about 25 kDa, is the mature, active form. Themature dimer appears to circulate in the blood as an inactive latentcomplex bound to the propeptide. As used herein the term “myostatinantagonist” defines a class of agents that inhibits or blocks at leastone activity of myostatin, or alternatively, blocks or reduces theexpression of myostatin or its receptor (for example, by interferencewith the binding of myostatin to its receptor and/or blocking signaltransduction resulting from the binding of myostatin to its receptor).Such agents therefore include agents which bind to myostatin itself orto its receptor.

Myostatin antagonists for use according to the disclosure includeantibodies to GDF-8; antibodies to GDF-8 receptors; soluble GDF-8receptors and fragments thereof (e.g., the ActRIIB fusion polypeptidesas described in U.S. Patent Publication Number 2004/0223966, which isincorporated herein by reference in its entirety, including solubleActRIIB receptors in which ActRIIB is joined to the Fc portion of animmunoglobulin); GDF-8 propeptide and modified forms thereof (e.g., asdescribed in WO 2002/068650 or U.S. Pat. No. 7,202,210, including formsin which GDF-8 propeptide is joined to the Fc portion of animmunoglobulin and/or form in which GDF-8 is mutated at an aspartate(asp) residue, e.g., asp-99 in murine GDF-8 propeptide and asp-100 inhuman GDF-8 propeptide); a small molecule inhibitor of GDF-8;follistatin (e.g., as described in U.S. Pat. No. 6,004,937, incorporatedherein by reference) or follistatin-domain-containing proteins (e.g.,GASP-1 or other proteins as described in U.S. Pat. No. 7,192,717 andU.S. Pat. No. 7,572,763, each incorporated herein by reference); andmodulators of metalloprotease activity that affect GDF-8 activation, asdescribed in U.S. Patent Publication Number 2004/0138118, incorporatedherein by reference.

Additional myostatin antagonists include myostatin antibodies which bindto and inhibit or neutralize myostatin (including the myostatinproprotein and/or mature protein, in monomeric or dimeric form).Myostatin antibodies are mammalian or non-mammalian derived antibodies,for example an IgNAR antibody derived from sharks, or humanizedantibodies, or comprise a functional fragment derived from antibodies.Such antibodies are described, for example, in WO 2005/094446 and WO2006/116269, the content of which is incorporated herein by reference.Myostatin antibodies also include those antibodies that bind to themyostatin proprotein and prevent cleavage into the mature active form.Additional antibody antagonists include the antibodies described in U.S.Pat. No. 6,096,506 and U.S. Pat. No. 6,468,535 (each of which isincorporated herein by reference). In some embodiments, the GDF-8inhibitor is a monoclonal antibody or a fragment thereof that blocksGDF-8 binding to its receptor. Further embodiments include murinemonoclonal antibody JA-16 (as described in U.S. Pat. No. 7,320,789 (ATCCDeposit No. PTA-4236); humanized derivatives thereof and fully humanmonoclonal anti-GDF-8 antibodies (e.g., Myo29, Myo28 and Myo22, ATCCDeposit Nos. PTA-4741, PTA-4740, and PTA-4739, respectively, orderivatives thereof) as described in U.S. Pat. No. 7,261,893 andincorporated herein by reference.

In still further embodiments, myostatin antagonists include solublereceptors which bind to myostatin and inhibit at least one activitythereof. The term “soluble receptor” herein includes truncated versionsor fragments of the myostatin receptor that specifically bind myostatinthereby blocking or inhibiting myostatin signal transduction. Truncatedversions of the myostatin receptor, for example, include the naturallyoccurring soluble domains, as well as variations produced by proteolysisof the N- or C-termini. The soluble domain includes all or part of theextracellular domain of the receptor, either alone or attached toadditional peptides or other moieties. Because myostatin binds activinreceptors (including the activin type IEB receptor (ActRHB) and activintype HA receptor (ActRHA)), activin receptors can form the basis ofsoluble receptor antagonists. Soluble receptor fusion proteins can alsobe used, including soluble receptor Fc (see U.S. Patent PublicationNumber 2004/0223966 and WO 2006/012627, both of which are incorporatedherein by reference in their entireties).

Other myostatin antagonists based on the myostatin receptors are ALK-5and/or ALK-7 inhibitors (see for example WO 2006/025988 and WO2005/084699, each incorporated herein by reference). As a TGF-βcytokine, myostatin signals through a family of single transmembraneserine/threonine kinase receptors. These receptors can be divided in twoclasses, the type I or activin-like kinase (ALK) receptors and type IIreceptors. The ALK receptors are distinguished from the Type IIreceptors in that the ALK receptors (a) lack the serine/threonine-richintracellular tail, (b) possess serine/threonine kinase domains that arehighly homologous among Type I receptors, and (c) share a commonsequence motif called the GS domain, consisting of a region rich inglycine and serine residues. The GS domain is at the amino terminal endof the intracellular kinase domain and is believed to be critical foractivation by the Type II receptor. Several studies have shown thatTGF-β signaling requires both the ALK (Type I) and Type II receptors.Specifically, the Type II receptor phosphorylates the GS domain of theType 1 receptor for TGFβ ALK5, in the presence of TGFβ. The ALK5, inturn, phosphorylates the cytoplasmic proteins smad2 and smad3 at twocarboxy terminal serines. Generally, it is believed that in manyspecies, the Type II receptors regulate cell proliferation and the TypeI receptors regulate matrix production. Various ALK5 receptor inhibitorshave been described (see, for example, U.S. Pat. No. 6,465,493, U.S.Pat. No. 6,906,089, U.S. Patent Publication Numbers 2003/0166633,2004/0063745 and 2004/0039198, the disclosures of which are incorporatedherein by reference). Thus, the myostatin antagonists for use accordingto the disclosure may comprise the myostatin binding domain of an ALK5and/or ALK7 receptor.

Other myostatin antagonists include soluble ligand antagonists thatcompete with myostatin for binding to myostatin receptors. The term“soluble ligand antagonist” herein refers to soluble peptides,polypeptides or peptidomimetics capable of non-productively binding themyostatin receptor(s) (e.g., the activin type HB receptor (ActRHA)) andthereby competitively blocking myostatin-receptor signal transduction.Soluble ligand antagonists include variants of myostatin, also referredto as “myostatin analogs” that have homology to, but not the activityof, myostatin. Such analogs include truncates (such as N- or C-terminaltruncations, substitutions, deletions, and other alterations in theamino acid sequence, such as variants having non-amino acidsubstitutions).

Additional myostatin antagonists contemplated by the disclosure includeinhibitory nucleic acids as described herein. These antagonists includeantisense or sense polynucleotides comprising a single-strandedpolynucleotide sequence (either RNA or DNA) capable of binding to targetmRNA (sense) or DNA (antisense) sequences. Thus, RNA interference (RNAi)produced by the introduction of specific small interfering RNA (siRNA),may also be used to inhibit or eliminate the activity of myostatin.

In specific embodiments, myostatin antagonists include, but are notlimited to, follistatin, the myostatin prodomain, growth anddifferentiation factor 11 (GDF-11) prodomain, prodomain fusion proteins,antagonistic antibodies or antibody fragments that bind to myostatin,antagonistic antibodies or antibody fragments that bind to the activintype IEB receptor, soluble activin type IHB receptor, soluble activintype IEB receptor fusion proteins, soluble myostatin analogs (solubleligands), polynucleotides, small molecules, peptidomimetics, andmyostatin binding agents. Other antagonists include the peptideimmunogens described in U.S. Pat. No. 6,369,201 and WO 2001/05820 (eachof which is incorporated herein by reference) and myostatin multimersand immunoconjugates capable of eliciting an immune response and therebyblocking myostatin activity. Other antagonists include the proteininhibitors of myostatin described in WO 2002/085306 (incorporated hereinby reference), which include the truncated Activin type II receptor, themyostatin pro-domain, and follistatin. Other myostatin inhibitorsinclude those released into culture from cells overexpressing myostatin(see WO 2000/43781), dominant negative myostatin proteins (see WO2001/53350) including the protein encoded by the Piedmontese allele, andmature myostatin peptides having a C-terminal truncation at a positioneither at or between amino acid positions 335 to 375. The small peptidesdescribed in U.S. Patent Publication Number 2004/0181033 (incorporatedherein by reference) that comprise the amino acid sequence WMCPP, arealso suitable for use in the compositions of the disclosure.

Vectors

An appropriate expression vector may be used to deliver exogenousnucleic acid to a recipient muscle cell in the methods of thedisclosure. In order to achieve effective gene therapy, the expressionvector must be designed for efficient cell uptake and gene productexpression. Use of adenovirus or adeno-associated virus (AAV) basedvectors for gene delivery have been described [Berkner, Current Topicsin Microbiol. and Imunol. 158: 39-66 (1992); Stratford-Perricaudet etal., Hum. Gene Ther. 1: 241-256 (1990); Rosenfeld et al., Cell 8:143-144 (1992); Stratford-Perricaudet et al., J. Clin. Invest. 90:626-630 (1992)]. In one specific embodiment, the adeno-associated virusvector is AAV9. Specific methods for gene therapy useful in the contextof the present disclosure depend largely upon the expression systememployed; however, most methods involve insertion of coding sequence atan appropriate position within the expression vector, and subsequentdelivery of the expression vector to the target muscle tissue forexpression.

Additional delivery systems useful in the practice of the methods of thedisclosure are discussed in U.S. Patent Publication Numbers 2012/0046345and 2012/0039806, each of which is incorporated herein by reference inits entirety.

Therapeutic Endpoints

In various aspects of the disclosure, use of the agent(s) and optionaladditional agent(s) as described herein provide one or more benefitsrelated to specific therapeutic endpoints relative to a patient notreceiving the agent(s) and/or additional agent(s).

In embodiments wherein the TGFβ-related disease is a muscle-relateddisease (e.g., a muscular dystrophy or cardiomyopathy), therapeuticendpoints include, but are not limited to, length of time until apatient is non-ambulatory, ambulatory capacity as measured by, forexample and without limitation, six-minute-walk distance which has beenshown to correlate with human LTBP4 SNPs [see, for example, Hersh etal., Am J Respir Crit Care Med. 173(9): 977-84 (2006)], relative healthof heart as determined by, e.g., echocardiography, magnetic resonanceimaging (MRI), muscle mechanics, pulmonary function and/or amount oftissue fibrosis.

With respect to the length of time until a patient is non-ambulatory, itis contemplated that, in some embodiments, a patient that isadministered one or more agent(s) and, optionally, additional agent(s)remains ambulatory at least 1 day and up to any of about 5, about 10,about 30, about 60 or about 90 days longer than a patient not sotreated. In further embodiments, a patient that is administered one ormore agent(s) and optional additional agent(s) remains ambulatory atleast about 1 month and up to any of about 2, about 4, about 6, about 8,about 10 or about 12 months longer than a patient not so treated. Stillfurther embodiments of the disclosure contemplate that a patient that isadministered one or more agent(s) and, optionally, additional agent(s)remains ambulatory at least about 1 year and up to any of about 1.5,about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10 or more years longer than a patient not so treated.

In embodiments wherein the TGFβ-related disease is a cancer, therapeuticendpoints include but are not limited to a reduction in tumor volume(i.e., the size of the tumor measured by the amount of space taken up byit expressed in traditional units of volume (e.g., cubic centimeters) oras a percentage of the tissue or organ within which it is found (e.g.,the tumor volume of prostate cancer is the percentage of the prostatetaken up by the tumor)) and/or a reduction in metastasis. With respectto the reduction in tumor volume and/or a reduction in metastasis, it iscontemplated that in some embodiments the tumor volume or amount ofmetastasis is reduced in a patient that is administered one or moreagent(s) and, optionally, additional agent(s) by about 1% relative to apatient not so treated. In further embodiments, the tumor volume oramount of metastasis is reduced in a patient that is administered one ormore agent(s) and, optionally, additional agent(s) by at least about 1%and up to any of about 2%, about 5%, about 10% or about 15% relative toa patient not so treated. In still further embodiments, the tumor volumeor amount of metastasis is reduced in a patient that is administered oneor more agent(s) and, optionally, additional agent(s) by at least about10% and up to about 15%, about 20%, about 25% or about 30% relative to apatient not so treated. In further embodiments, the tumor volume oramount of metastasis is reduced in a patient that is administered one ormore agent(s) and, optionally, additional agent(s) by at least about 10%and up to any of about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, about 99% or more relative to a patient not sotreated. In specific embodiments, the tumor volume or amount ofmetastasis is reduced in a patient that is administered one or moreagent(s) and, optionally, additional agent(s) by at least about 1%,about 2%, about 5%, about 10%, about 20%, about 25%, about 30%, about35%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 99% or more relative to a patient not so treated.Methods of measuring tumor volume as well as amount of metastasis areknown in the art.

In embodiments wherein the TGFβ-related disease is a viral disease,therapeutic endopoints relate to the viral load in the patient. Methodsof determining viral load are well known in the art and can bequantitated using methods such as polymerase chain reaction (PCR),reverse-transcriptase PCR (RT-PCR), probe-specific amplification or bythe branched DNA (bDNA) method. In various embodiments, the viral loadof a patient being administered one or more agent(s) and, optionally,additional agent(s) of the disclosure is reduced by at least about 1%and up to any of about 5%, about 10%, about 20%, about 30%, about 40% orabout 50% relative to a patient not so treated. In further embodiments,the viral load of a patient being administered one or more agent(s) and,optionally, additional agent(s) of the disclosure is reduced by at leastabout 10% and up to any of about 20%, about 50%, about 70%, about 80%,about 90%, about 99% or more relative to a patient not so treated. Inspecific embodiments, the viral load of a patient being administered oneor more agent(s) and/or additional agent(s) of the disclosure is reducedby at least about 1%, about 2%, about 5%, about 10%, about 20%, about25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 99% or more relative to a patientnot so treated.

In general, a therapeutic endpoint achieved by practice of the methodsof the disclosure is a reduction in the amount of fibrosis in a patientbeing administered one or more agent(s) and, optionally, additionalagent(s) of the disclosure. Relative amounts of fibrosis in a patientcan be quantitated by tissue biopsy and subsequent histology, e.g., byquantifying Evans blue dye uptake as a measure of myofiber or cellulardamage [Heydemann et al., Neuromuscular Disorders 15(9-10): 601-9(2005)], and/or quantitation of hydroxyproline content as describedpreviously [Swaggart et al., Physiol Genomics 43: 24-31 (2011)]. Invarious embodiments, the amount of fibrosis in a patient beingadministered one or more agent(s) and, optionally, additional agent(s)of the disclosure is reduced by at least about 1% and up to any of about5%, about 10%, about 20%, about 30%, about 40% or about 50% relative toa patient not so treated. In further embodiments, the amount of fibrosisin a patient being administered one or more agent(s) and, optionally,additional agent(s) of the disclosure is reduced by at least about 10%and up to about 20%, about 50%, about 70%, about 80%, about 90%, about99% or more relative to a patient not so treated. In specificembodiments, the amount of fibrosis in a patient being administered oneor more agent(s) and/or additional agent(s) of the disclosure is reducedby at least about 1%, about 2%, about 5%, about 10%, about 20%, about25%, about 30%, about 35%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 99% or more relative to a patientnot so treated.

The amount of fibrosis in a patient can be routinely determined by oneof ordinary skill in the art. For example, and without limitation, theamount of fibrosis can be determined by taking a muscle biopsy from apatient, sectioning the muscle onto slides and assessing the amount offibrosis as revealed by staining techniques known in the art (e.g.,Hematoxylin and Eosin (H&E) staining and/or Masson's trichromestaining). Alternatively, or in addition, the amount of fibrosis can bedetermined in vivo by using magnetic resonance imaging (MRI).

Dosing/Administration/Kits

A particular administration regimen for a particular subject willdepend, in part, upon the agent and optional additional agent used, theamount of the agent and optional additional agent administered, theroute of administration, the particular ailment being treated, and thecause and extent of any side effects. The amount of agent and optionaladditional agent administered to a subject (e.g., a mammal, such as ahuman) is sufficient to effect the desired response. Dosage typicallydepends upon a variety of factors, including the particular agent and/oradditional agent employed, the age and body weight of the subject, aswell as the existence and severity of any disease or disorder in thesubject. The size of the dose also will be determined by the route,timing, and frequency of administration.

Accordingly, the clinician may titer the dosage and modify the route ofadministration to obtain optimal therapeutic effect, and conventionalrange-finding techniques are known to those of ordinary skill in theart. Purely by way of illustration, in some embodiments, the methodcomprises administering, e.g., from about 0.1 μg/kg up to about 100mg/kg or more, depending on the factors mentioned above. In otherembodiments, the dosage may range from 1 μg/kg up to about 75 mg/kg; or5 μg/kg up to about 50 mg/kg; or 10 μg/kg up to about 20 mg/kg. Incertain embodiments, the dose comprises about 0.5 mg/kg to about 20mg/kg (e.g., about 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.3 mg/kg, 2.5 mg/kg, 3mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg) of agent and optionaladditional agent. In embodiments in which an agent and additional agentare administered, the above dosages are contemplated to represent theamount of each agent administered, or in further embodiments the dosagerepresents the total dosage administered. Given the chronic nature ofmany TGFβ-related disorders, it is envisioned that a subject willreceive the agent and/or additional agent over a treatment courselasting weeks, months, or years, and may require one or more doses dailyor weekly. Dosages are also contemplated for once daily, twice daily(BID) or three times daily (TID) dosing. A unit dose may be formulatedin either capsule or tablet form. In other embodiments, the agent andoptional additional agent is administered to treat an acute condition(e.g., acute muscle injury or acute myocardial injury) for a relativelyshort treatment period, e.g., one to 14 days.

Suitable methods of administering a physiologically-acceptablecomposition, such as a pharmaceutical composition comprising an agentand optional additional agent described herein, are well known in theart. Although more than one route can be used to administer an agentand/or additional agent, a particular route can provide a more immediateand more effective avenue than another route. Depending on thecircumstances, a pharmaceutical composition is applied or instilled intobody cavities, absorbed through the skin or mucous membranes, ingested,inhaled, and/or introduced into circulation. In some embodiments, acomposition comprising an agent and/or additional agent is administeredintravenously, intraarterially, or intraperitoneally to introduce anagent and optional additional agent into circulation. Non-intravenousadministration also is appropriate, particularly with respect to lowmolecular weight therapeutics. In certain circumstances, it is desirableto deliver a pharmaceutical composition comprising the agent and/oradditional agent orally, topically, sublingually, vaginally, rectally;through injection by intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraportal,intralesional, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intranasal, urethral, or enteral means; bysustained release systems; or by implantation devices. If desired, theagent and/or additional agent is administered regionally viaintraarterial or intravenous administration to a region of interest,e.g., via the femoral artery for delivery to the leg. In one embodiment,the composition is administered via implantation of a membrane, sponge,or another appropriate material within or upon which the desired agentand optional additional agent has been absorbed or encapsulated. Wherean implantation device is used, the device in one aspect is implantedinto any suitable tissue, and delivery of the desired agent and/oradditional agent is, in various embodiments, effected via diffusion,time-release bolus, or continuous administration. In other embodiments,the agent and optional additional agent is administered directly toexposed tissue during surgical procedures or treatment of injury, or isadministered via transfusion of blood products. Therapeutic deliveryapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,399,363.

In some embodiments facilitating administration, the agent and optionaladditional agent in one embodiment is formulated into aphysiologically-acceptable composition comprising a carrier (i.e.,vehicle, adjuvant, buffer, or diluent). The particular carrier employedis limited only by chemico-physical considerations, such as solubilityand lack of reactivity with the agent and/or additional agent, by theroute of administration, and by the requirement of compatibility withthe recipient organism. Physiologically acceptable carriers are wellknown in the art. Illustrative pharmaceutical forms suitable forinjectable use include, without limitation, sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (for example, see U.S. Pat.No. 5,466,468). Injectable formulations are further described in, e.g.,Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia.Pa., Banker and Chalmers. eds., pages 238-250 (1982), and ASHP Handbookon Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986),incorporated herein by reference).

A pharmaceutical composition comprising an agent and optional additionalagent as provided herein is optionally placed within containers/kits,along with packaging material that provides instructions regarding theuse of such pharmaceutical compositions. Generally, such instructionsinclude a tangible expression describing the reagent concentration, aswell as, in certain embodiments, relative amounts of excipientingredients or diluents that may be necessary to reconstitute thepharmaceutical composition.

The disclosure thus includes administering to a subject one or moreagent(s), in combination with one or more additional agent(s), eachbeing administered according to a regimen suitable for that medicament.Administration strategies include concurrent administration (i.e.,substantially simultaneous administration) and non-concurrentadministration (i.e., administration at different times, in any order,whether overlapping or not) of the agent and one or more additionalagents(s). It will be appreciated that different components areoptionally administered in the same or in separate compositions, and bythe same or different routes of administration.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. In addition, the entiredocument is intended to be related as a unified disclosure, and itshould be understood that all combinations of features described hereinare contemplated, even if the combination of features are not foundtogether in the same sentence, or paragraph, or section of thisdocument. For example, where protein therapy is described, embodimentsinvolving polynucleotide therapy (using polynucleotides/vectors thatencode the protein) are specifically contemplated, and the reverse alsois true. With respect to elements described as one or more members of aset, it should be understood that all combinations within the set arecontemplated.

EXAMPLES Example 1 Structure-Function Relationship Between theProline-Rich Region of LTBP4 and Proteolytic Susceptibility

The data provided in this Example show that the proline-rich region ofLTBP4 contributes to its proteolytic susceptibility.

LTBP4 binds to TGFβ in the extracellular matrix (ECM), where it servesas a readily available TGFβ storage site. A 36-nucleotide deletion wasidentified in the proline-rich domain of murine LTBP4 that associateswith enhanced pathogenic features of muscular dystrophy in mice. Thisregion in murine LTBP4 is associated with variable susceptibility toproteolysis. Sequence comparison analysis between LTBP4 from mouse andhumans reveals an even larger deletion in the proline-rich region ofhuman LTBP4. Thus, consistent with the murine deletion being associatedwith pathogenic features and variable proteolysis [see Heydemann et al.,J Clin Invest. 119(12): 3703-12 (2009)], it was contemplated that thelarger deletion of the proline-rich region of human LTBP4 was associatedwith enhanced susceptibility to proteolytic cleavage.

To investigate this possibility, a portion of the human LTBP4 codingregion was ligated into an expression vector to express the proline-richregion. The TP fragment (amino acids 483-565 of the human LTBP4 protein(SEQ ID NO: 1)) was expressed and migrated as a 3.5 KDa protein althoughits predicted molecular mass is 8.9 KDa. A second fragment, TP2Efragment (amino acids 357-586 of the human LTBP4 protein (SEQ ID NO: 1))was also expressed. Its predicted molecular mass is 24.5 KDa, yet itelectrophoretically migrated as a 31 KDa protein. TP2E included the twoEGF-like domains that flank the proline-rich region of LTBP4 along withthe amino terminal 8-cysteine rich region immediately amino-terminal ofthe proline-rich region. Murine TP2E and TP each contained an additional44 amino acids compared to the human sequences, reflecting the largerproline-rich region. The murine TP2E electrophoretically migrates as a35 KDa protein while its calculated molecular mass is 30.58 KDa.

Susceptibility to Proteolysis In Vitro

Human and mouse TP2E fragments were expressed in vitro using atranscription-translation coupled assay (Promega TnT® Quick Coupled invitro Transcription/Translation System) and the expressed fragments werelabeled using ³⁵S-Cysteine. Dose-response and time course experimentswere performed with elastase and plasmin, which are both serineproteases that cleave LTBP4, to determine the differential digestion ofthe human and mouse TP2E fragments. Data from these experiments showedthat the human TP2E fragment is more readily cleaved than the mouseLTBP4 sequence (FIG. 3).

Effects of Smaller Fragments of LTBP4 on TGFβ Signaling

An antibody to the proline-rich region of human LTBP4 was generated toinhibit LTBP4 cleavage. This antibody was tested and confirmed torecognize and bind to the full-length human LTBP4 by immunoblot.Conditions were then optimized for the digestion of full-length humanLTBP4 by plasmin, and inhibition of proteolysis using the antibody wastested. The data in FIG. 4 show that the anti-LTBP4 antibodyspecifically inhibited the protein digestion compared to a nonrelatedantibody raised in the same species (FIG. 4).

Example 2 Effect of Human LTBP4 Expression on Muscle and CardiacPhenotypes

LTBP4 plays a critical role in TGFβ secretion and activation in cardiacmuscle, skeletal muscle and lung. Human LTBP4 has a larger deletion inthe proline-rich region compared to a mutant murine LTBP4, withwild-type murine LTBP4 used as a reference. Thus, it is contemplatedthat human LTBP4 is associated with increased pathogenic TGFβ signalingand, therefore, will be associated with more severe disease in mice withmuscular dystrophy.

Transgenic Mice Expressing Human LTBP4

A mouse harboring the human LTBP4 gene was generated according tostandard protocols [see, e.g., Heintz, Nat Rev Neurosci. 2(12):861-70(2001)]. A bacterial artificial chromosome (BAC) that included thecomplete human LTBP4 gene; the BAC transgenic-positive (Tg+) mice arereferred to as hLTBP4 Tg+. To generate the hLTBP4 Tg+mice, a single,unmodified BAC clone (clone number CDT-2166J9) was used to inject afertilized oocyte using conventional methodology [see, e.g., Heintz, NatRev Neurosci. 2(12):861-70 (2001)]. The human sequence of this BAC(Genbank accession number AC010412.9; SEQ ID NO: 7) contains 155085 bpfrom chromosome 19. The LTBP4 gene spans from 19891 to 57891 bp of thisclone. Eleven founder lines were evaluated by PCR and found to containthe full-length human LTBP4, including promoter regions. Six lines werechosen for breeding to ensure that these mice were passing the BAC intheir germline. By RT-PCR, it was determined that the human LTBP4 mRNAwas expressed in cardiac and skeletal muscle of the transgenic mice. Atpresent, there is no antibody that distinguishes human LTBP4 from mouseLTBP4; human and mouse LTBP4 are 98% similar. Overall, LTBP4 expressionmay be slightly elevated in hLTBP4 Tg+ mice compared to littermatecontrols. hLTBP4 Tg+ mice are outwardly normal and breed normally.Histological examination showed grossly normal histology in brain,kidney, lung, heart and muscle. Interestingly, hLTBP4 Tg+ skeletalmuscle fibers were significantly larger than littermate controltransgene negative mice. It is contemplated that even modestoverexpression of LTBP4 may be sufficient to bind other TGFβ superfamilymembers such as myostatin, and sequestration of myostatin would inhibitmyostatin activity and would be expected to result in larger musclefibers.

The hLTBP4 Tg+ animal will be bred to the mouse mdx model of DuchenneMuscular Dystrophy and the phenotype and TGFβ signaling capacity will beassessed. Ten mice of each genotype (hLTBP4 Tg+/mdx, mdx, hLTBP4+ andWT) will be generated. Basic neuromuscular function will be evaluatedusing SHIRPA protocols. SHIRPA is a combination of neurological teststhat assess neuromuscular function [Rafael et al., Mamm Genome. 11(9):725-8 (2000)]. For example and without limitation, grip strength,running capacity, wire maneuver and rotorod are basic tests that will beused to assess muscle function. In addition, cardiac function will beassessed using echocardiography, and histology will be performed toevaluate fibrosis and membrane permeability using Evans blue dye uptake.All analyses will be conducted on male mice at 8 weeks of age. A cohortof mice will also be aged to examine the effect on mice at a later timepoint(s). Fibroblasts will also be isolated from these mice to determinetheir level of SMAD signaling using methods as previously described[Heydemann et al., J Clin Invest. 119(12): 3703-12 (2009)]. It isexpected that insertion of the human LTBP4 will result in increased SMADsignaling and enhancement of the mdx phenotype.

Example 3 LTBP4 Peptides and Antibody Generation

Antibodies were generated using multiple different peptides includingthe mouse and human LTBP4 sequences (see Table 1, below). Each of thepeptides in Table 1 cross reacts to the human protein as determined byimmunoblotting. A longer LTBP4 peptide,FLPTHRLEPRPEPRPDPRPGPELPLPSIPAWTGPEIPESGPSS (SEQ ID NO: 6), is alsocontemplated for use according to the disclosure. Humanized monoclonalantibodies directed against LTBP4 will also be generated.

TABLE 1 LTBP4 peptides used for antibody generation. ″Species″indicates antibody source. Western Antibody Antigen Blot Results on NameSpecies Peptide Used Source Activity Proteolysis mLTBP4d36- chickenEPRPRPEPRPQPESQPWP Mouse-D2 +++ NA 829 (SEQ ID NO: 2) mLTBP4d36- chickenEPRPRPEPRPQPESQPWP Mouse-D2 ++ NA 830 (SEQ ID NO: 2) hLTBP4pr- chickenEPRPEPRPDPRPGPELP Human ++++ positive 831 (SEQ ID NO: 3) hLTBP4pr-chicken EPRPEPRPDPRPGPELP Human ++ NA 832 (SEQ ID NO: 3) mLTBP4ins-rabbit ESQPRPESRPRPESQPWP Mouse-129 ++ NA 24226 (SEQ ID NO: 4)mLTBP4ins- rabbit ESQPRPESRPRPESQPWP Mouse-129 + NA 24226 (SEQ ID NO: 4)hLTBP4(511- rabbit PERPEPRPDPRPGPELPLP Human NA NA 530) (SEQ ID NO: 5)28200 hLTBP4(511- rabbit PERPEPRPDPRPGPELPLP Human NA NA 530)(SEQ ID NO: 5) 28199

Table 1 shows that each antibody recognized a protein the size of humanLTBP4, as determined by immunoblot. The data in the table also indicatesthat the antibody raised against the human sequence (SEQ ID NO: 3)protects LTBP4 against proteolysis in vitro (FIG. 6, described below)and, given the cross reactivity, the other anti-hLTBP4 antibodies arealso expected to protect hLTBP4 from proteolysis. Enzyme-linkedimmunosorbent assays (ELISA) will also be performed to compare therelative affinity of antibodies to each peptide using serum and purifiedantibodies.

Proteolysis of LTBP4 can be Inhibited with LTBP4 Antibodies

It was contemplated that the insertion/deletion polymorphism in murineLtbp4 discussed hereinabove indicated that the proline-rich region isimportant since the presence or absence of 12 additional amino acids inthis region explains its ability to reduce membrane leak and suppressfibrosis, two activities that were attributed to LTBP4's ability tosequester TGFβ. This position is consistent with the differentialsensitivity to proteolysis of the various forms of LTBP4 and theassociated TGFβ activity in the form of nuclear pSMAD [Heydemann et al.,J Clin Invest. 119: 3703-12 (2009)].

To demonstrate that the proline-rich region of human LTBP4 wassusceptible to proteolysis, protein domains were expressed using invitro transcription and translation according to methods as previouslydescribed [Heydemann et al., J Clin Invest. 119(12): 3703-12 (2009)]. Bydesign, only the carboxy-terminus of these expressed proteins waslabeled. The expressed fragments were exposed to plasmin. Murine LTBP4with the 12-amino-acid insertion was largely resistant to proteolysiswhile the murine LTBP4 deleted for the 12 amino acids was readilydegraded (FIG. 5, middle and right lanes). The human LTBP4 was mostreadily degraded (FIG. 5, left lanes). Similar results were obtainedwith elastase. It is contemplated that this region (i.e., the regionincluded in the TP and TP2E sequences) is a general serine proteasetarget. Antibodies were generated that were directed at the proline-richregion and it was found that these antibodies inhibited LTBP4 cleavagein vitro (FIG. 6). A nonspecific antibody generated from the samespecies showed no blocking effect. Additional anti-LTBP4 antibodies havebeen generated, and Fab fragments will be purified and tested becausethese fragments are expected to be more useful for in vivo delivery.

Full-length LTBP4 protein, produced from cultured cells, is alsosusceptible to plasmin proteolysis (FIG. 6). With muscle injury, such asthe injury that occurs in DMD, release of proteases into theextracellular matrix is expected to result in LTBP4 cleavage. Thesources of these proteases in vivo may be inflammatory cells,fibroblasts or the myofibers. Increased LTBP4 cleavage was shown tocorrelate with increased fibrosis, increased muscle membrane leak,increased muscle weakness and increased TGFβ signaling [Heydemann etal., J Clin Invest. 119(12): 3703-12 (2009)]. Reduction of TGFβsignaling was shown to improve outcome in muscular dystrophy [Cohn etal., Nat Med. 13(2): 204-10 (2007); Goldstein et al., Hum Mol Genet.20(5): 894-904 (2011)].

This example demonstrates that proteolysis of the proline-rich region ofLTBP4 can be inhibited by antibodies provided herein.

Example 4 Transgenic Mice Harboring Human Ltbp4

A human bacterial artificial chromosome (BAC) carrying the full lengthhuman LTBP4 gene was isolated and characterized. This BAC was injectedinto mice and several lines of transgenic mice were characterized. HumanLTBP4 (hLTBP4-BAC) transgenic mice were bred to mdx mice. The humanLTBP4 BAC in the normal background resulted in larger myofiber diameter,a sign of hypertrophy. When the human LTBP4 BAC was in the mdxbackground, it resulted in enhanced fibrosis in skeletal and cardiacmuscle as well as reduced grip strength, relative to control mice thatdid not carry the transgene (FIG. 7). This supports the observation thatthe human LTBP4 sequence, with its larger deletion in the proline-richregion, enhances the muscular dystrophy phenotype. These mice will befurther used to test whether antibodies directed against human LTBP4 canreduce muscular dystrophy fibrosis and muscle membrane leakage.

Example 5 In Vivo Studies

Short-term studies are conducted in dystrophic mice (i.e., mdx and limbgirdle muscular dystrophy (LGMD)) to determine safety and efficacy ofinhibiting LTBP4 cleavage in vivo. Animals are treated from 3 weeks to 8weeks of age with antibody injections, three times weekly, delivered viaintraperitoneal injection. Dose responsiveness is determined.Echocardiography, plethysmography, muscle harvest and ex vivo musclemechanics are conducted on treated animals and controls. Target tissuesare studied, including heart, diaphragm, quadriceps, gluteus/hamstrings,gastrocnemius/soleus, triceps and abdominal muscles, according topreviously identified protocols [Heydemann et al., Neuromuscul Disord.15: 601-9 (2005); Heydemann, et al., J Clin Invest. 119: 3703-12 (2009);Swaggart et al., Physiol Genomics. 43: 24-31 (2011)]. TGFβ signaling isalso determined.

Long-term studies are conducted in dystrophic mice to determine thesafety and efficacy of the treatment. Once dosing has been determined,cohorts of mice are treated from 3 weeks until 1 year of age. A similaranalysis of efficacy are undertaken, as discussed above (i.e.,echocardiography, plethysmography, muscle harvest and ex vivo musclemechanics). Analysis of other organs, including lung, colon, kidney,brain, and other tissues, is included. Mice that are null for LTBP4develop cardiomyopathy, pulmonary fibrosis and colon cancer. BecauseLTBP4 is not ablated in these studies, these cardiomyopathy, pulmonaryfibrosis and colon cancer defects are not expected, consistent with theresults of the genetic studies described above. Nonetheless, off-targettissues are also analyzed.

The studies described above are expected to show that inhibition ofLTBP4 cleavage in vivo results in decreased TGFβ signaling, which isfurther expected to lead to a decrease in membrane permeability as wellas a decrease in fibrosis in the muscles of dystrophic mice. Theseresults will be evidenced by an improvement or lack of decline intherapeutic endpoints as described herein, thereby establishing thatblockage of LTBP4 proteolysis is a robust therapeutic in the treatmentof TGFβ superfamily protein-related diseases.

Example 6 LTBP4 Interacts with Myostatin In Vitro

The ability of LTBP4 to directly interact with myostatin, a TGF-βsuperfamily member, was also investigated. The methods used toinvestigate the interaction were as follows. Full length LTBP4 wascloned into an expression vector (pcDNA3.1, Life Technologies(Invitrogen), Grand Island, N.Y.) and the Xpress epitope tag (LifeTechnologies (Invitrogen), Grand Island, N.Y.) was added to its 5′end/amino terminus. Full-length myostatin, encoding the propeptide andmature regions, was tagged at its 3′ end/carboxy terminus with the mycepitope tag. Both plasmids were introduced into HEK293 (Human EmbryonicKidney 293) cells. The cells were lysed and the proteins were blotted orimmunoprecipitated with either antibody 28200 or, in a separateexperiment, antibody 28199 (see Table 1). Both of these rabbitpolyclonal antibodies are directed at the LTBP4 proline-rich region. Theimmunoprecipitated material was then blotted with the anti-myc antibody,showing that myostatin associates with LTBP4 (FIG. 8).

This Example shows that LTBP4 is able to directly interact withmyostatin. The results indicate that, by inhibiting the proteolysis ofLTBP4 according to the present disclosure, one can sequester myostatinand prevent its activation and resultant downstream signaling. Becausemyostatin is a known negative regulator of muscle growth, the inhibitionof myostatin signaling is expected to result in increased muscle growthand increased muscle strength.

Example 7 Expression of Human LTBP4 in Mice Leads to Enhanced Damageafter Cardiotoxin Injury

Mice were generated to express the human LTBP4 gene on a bacterialartificial chromosome, and these transgenic mice were referred to ashLTBP4 Tg+ mice. The human LTBP4 protein is more readily proteolyzedbecause of its shorter proline-rich region. This increased proteolysisleads to enhanced damage in muscle due to increased TGFβ release.Cardiotoxin was injected into the tibialis anterior muscle of normal (WTw/CTX) and hLTBP4 transgenic mice (hLTBP4 Tg+ w/CTX). Transgenic micedisplayed enhanced injury after cardiotoxin injury seen as greaterinflammatory mononuclear cell infiltrate and fibrosis and fat depositioninto the injured muscle (FIG. 9A), similar to what is seen in musculardystrophy.

Normal (WT) and hLTBP4 muscle were injected with cardiotoxin to induceinjury. Immunoblotting with an anti-LTBP4 antibody showed increasedlevels of LTBP4 protein induced by injury in both normal and in hLTBP4transgenic muscle (FIG. 9B). hLTBP4 muscle was also found to beassociated with increased TGFβ signaling seen as nuclear localizedphosphorylated SMAD.

The results showed that expression of human LTBP4 protein in muscleleads to enhanced muscle damage following cardiotoxin injury.

Example 8 Anti-LTBP4 Antibodies Mitigate Muscle Injury In Vivo

To test whether anti-LTBP4 antibody mitigated skeletal muscle injury inmuscular dystrophy, experiments were carried out using hLTBP4/mdx mice.Cardiotoxin, which is known to cause necrosis of skeletal muscle cells,was injected into the tibialis anterior muscle to induce enhancedinjury. This injury model resolves within 2 weeks because a low-volumeinjection of 10 μl is used. hLTBP4/mdx mice (8 weeks of age) werepretreated on day 0 with either (i) PBS or (ii) antibody to LTBP4-831antibody at 5 mg/Kg intraperitoneally. On day 1, cardiotoxin wasinjected into the tibialis anterior muscle. LTBP4-831 antibody wasinjected on days 1, 3, and 5, each time delivering a 5 mg/Kg doseintraperitoneally. Mice were sacrificed on day 7 and tibialis anteriormuscle was harvested for study. The experimental design of sacrificingthe mice on day 7 was used because the LTBP4-831 antibody is a chickenantibody that was expected to be recognized as foreign after 2-3 weeks.

Following harvest, the muscle was processed for analysis bysnap-freezing in liquid nitrogen-cooled isopentane. The frozen musclewas sectioned and the sections were subjected to hematoxylin and eosin(H&E) staining.

Results of the experiment showed that, compared to PBS-injected mice,LTBP4-831 antibody-treated mice showed reduced central nucleation andreduced fibrosis (FIGS. 10A and 10B). Centralized nuclei are indicativeof newly formed (i.e., regenerating) myofibers, and reduced centralnucleation in the muscle of animals that were administered LTBP4-831antibody provides evidence that the antibody mitigated muscle injury inthe mice.

Example 9 Increased Inflammatory Infiltrate in hLTBP4/Mdx Mice Comparedto Mdx Mice

Quadriceps muscles obtained from both mdx and hLTPB4/mdx mice werestained with F4/80 antibodies that recognize and bind to activatedmacrophages (shown as speckles throughout the muscle). Theimmunofluorescent staining showed an increase in activated macrophagesin hLTBP4/mdx muscle compared to mdx muscle (FIG. 11A). hLTBP4/mdxmuscle showed an increase in cleaved LTBP4 protein compared to mdx,while little LTBP4 protein was seen in wild-type and hLTBP4 muscle inthe absence of injury or muscular dystrophy (FIG. 11B).

The results showed that there is an increase in inflammatory cellinfiltrate in the muscle of hLTBP4/mdx mice versus mdx mice. The resultsalso showed that hLTBP4/mdx muscle possessed increased cleaved LTBP4protein relative to mdx muscle.

The disclosed subject matter has been described with reference tovarious specific and preferred embodiments and techniques. It should beunderstood, however, that many variations and modifications may be madewhile remaining within the spirit and scope of the disclosed subjectmatter. All references cited herein are hereby incorporated by referencein their entireties, or to the extent that they provide relevantdisclosure, as would be ascertained from context.

What is claimed is:
 1. A method of treating a patient having atransforming growth factor beta (TGFβ) superfamily protein-relateddisease, comprising administering a therapeutically effective amount ofan agent that modulates proteolysis of latent TGFβ binding protein 4(LTBP4) to a patient in need thereof.
 2. A method of delaying onset orpreventing a transforming growth factor beta (TGFβ) superfamilyprotein-related disease, comprising administering an effective amount ofan agent that modulates proteolysis of latent TGFβ binding protein 4(LTBP4) to a patient in need thereof.
 3. The method of claim 1 or claim2 wherein the patient suffers from a disease selected from the groupconsisting of Duchenne Muscular Dystrophy, Limb Girdle MuscularDystrophy, Becker Muscular Dystrophy, myopathy, cystic fibrosis,pulmonary fibrosis, cardiomyopathy, acute lung injury, acute muscleinjury, acute myocardial injury, radiation-induced injury and coloncancer.
 4. The method of any one of claims 1-3 wherein the agent isselected from the group consisting of an anti-LTBP4 antibody and apeptide.
 5. The method of any one of claims 1-4 further comprisingadministering an effective amount of a second agent, wherein the secondagent is selected from the group consisting of a modulator of aninflammatory response, a promoter of muscle growth, a chemotherapeuticagent, and a modulator of fibrosis.
 6. A method of treating a patienthaving a transforming growth factor beta (TGFβ) superfamilyprotein-related disease, comprising administering to the patient atherapeutically effective amount of an agent that upregulates theactivity of latent TGFβ binding protein 4 (LTBP4).
 7. A method ofdelaying onset or preventing a transforming growth factor beta (TGFβ)superfamily protein-related disease, comprising administering to thepatient an effective amount of an agent that upregulates the activity oflatent TGFβ binding protein 4 (LTBP4).
 8. The method of claim 6 or claim7 wherein LTBP4 interacts with a TGFβ superfamily protein.
 9. The methodof any one of claim 8 wherein the TGFβ superfamily protein is selectedfrom the group consisting of TGFβ, a growth and differentiation factor(GDF), activin, inhibin, and a bone morphogenetic protein.
 10. Themethod of claim 9 wherein the GDF is myostatin.
 11. The method of anyone of claims 6-10 wherein the agent is selected from the groupconsisting of a peptide, an antibody and a polynucleotide capable ofexpressing a protein having LTBP4 activity.
 12. The method of claim 11wherein the agent is the peptide of claim
 21. 13. The method of claim 11wherein the agent is the antibody of claim
 20. 14. The method of claim11 wherein the polynucleotide is contained in a vector.
 15. The methodof claim 14 wherein the vector is a viral vector.
 16. The method ofclaim 15 wherein the viral vector is selected from the group consistingof a herpes virus vector, an adeno-associated virus (AAV) vector, anadeno virus vector, and a lentiviral vector.
 17. The method of claim 16wherein the AAV vector is recombinant AAV9.
 18. The method of any one ofclaims 6-17 wherein the patient has a disease selected from the groupconsisting of Duchenne Muscular Dystrophy, Limb Girdle MuscularDystrophy, Becker Muscular Dystrophy, myopathy, cystic fibrosis,pulmonary fibrosis, cardiomyopathy, acute lung injury, acute muscleinjury, acute myocardial injury, radiation-induced injury, and coloncancer.
 19. The method of any one of claims 6-18 further comprisingadministering an effective amount of a second agent, wherein the secondagent is selected from the group consisting of a modulator of aninflammatory response, a promoter of muscle growth, a chemotherapeuticagent and a modulator of fibrosis.
 20. An isolated antibody thatspecifically binds to a peptide comprising the sequence set forth in SEQID NO:
 5. 21. A peptide comprising the sequence as set out in any one ofSEQ ID NOs: 2-5, or a peptide that is at least 70% identical to thesequence as set out in SEQ ID NOs: 2-5 that retains an ability to act asa substrate for a protease.
 22. A pharmaceutical formulation comprisingan effective amount of the antibody of claim 20 or the peptide of claim21, and a pharmaceutically acceptable carrier or diluent.
 23. A kitcomprising a therapeutically effective amount of the antibody of claim20 or the peptide of claim 21, a pharmaceutically acceptable carrier ordiluent and instructions for use.
 24. The formulation of claim 22 or thekit of claim 23, further comprising an effective amount of a secondagent, wherein the second agent is selected from the group consisting ofa modulator of an inflammatory response, a promoter of muscle growth, achemotherapeutic agent and a modulator of fibrosis.